Increasing plant oil content by altering a negative regulator of acetyl-coa carboxylase

ABSTRACT

The present invention provides a method and means to change fatty acid and ultimately triacylglycerol production in plants and algae. Methods of the invention comprise the step of altering the activity levels of the committed step for de novo fatty acid biosynthesis, acetyl-CoA carboxylases (ACCase). More specifically, methods of the invention directly enhance the activity of ACCase by down-regulating the biotin/lipoyl attachment domain containing (BADC) genes through biotechnology or selective breeding approaches.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a 371 National Stage application of InternationalApplication No. PCT/US2016/041386, filed Jul. 7, 2016, which claims thebenefit of U.S. Provisional Application No. 62/211,371, filed Aug. 28,2015, the entire contents of which are herein incorporated by referencein its entirety.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH

This invention was made with Government support under Grant No. PGRP10S-1339385 awarded by National Science Foundation and Grant No. T32GM008396 by the National Institutes of Health. The Government hascertain rights in the invention.

INCORPORATION OF SEQUENCE LISTING

The sequence listing that is contained in the file named“UVMO115WO_ST25.txt,” which is 331 kilobytes as measured in MicrosoftWindows operating system and was created on Jul. 6, 2016, is filedelectronically herewith and incorporated herein by reference.

FIELD OF THE INVENTION

The present invention relates to methods and systems for increasingplant oil content, and more particularly to constructs and methods todown-regulate expression of a negative regulator of de novo fatty acidsynthesis in order to increase fatty acid and ultimately triacylglycerolproduction in plants and algae that harbor this family of negativeregulators.

BACKGROUND OF THE INVENTION

Vegetable oils are an important renewable source of hydrocarbons forfood, energy, and industrial feedstocks. As demand for this commodityincreases, discovering ways to enhance oil production in crops will bean agronomic priority. Oil production begins with the de novo fatty acidsynthesis (FAS) pathway to generate the acyl chains that are eventuallyesterified to glycerol to produce triacylglycerol, the major storagelipid in the seed. The committed step of de novo FAS is catalyzed byacetyl-coenzyme A carboxylase (ACCase) which carboxylates acetyl-CoA toform malonyl-CoA in a two-step reaction requiring ATP, bicarbonate, andbiotin cofactor. In prokaryotes, and in plastids of most plants, ACCaseis a heteromeric complex requiring four distinct subunits: biotincarboxylase (BC), biotin carboxyl carrier protein (BCCP), and α- andβ-carboxyltransferases (CT). Graminaceous monocots possess a homomericform of plastid ACCase where the catalytic components are adjoined intandem as a single polypeptide. Structural models for the heteromericACCase are primarily based on studies in Escherichia coli. The E. coliACCase is composed of two subcomplexes: an α/β-CT heterotetramer and aBC/BCCP heterotetramer. The components of the two subcomplexes formstable associations, while the subcomplexes themselves show a relativelyweak interaction with one another. This property has contributed to thedifficulties in biochemical and structural characterization ofheteromeric ACCase from plants. Plastidial ACCase is regulated by light,feedback inhibition, and a 2-oxoglutarate-binding protein PII. Itremains unknown if such regulation is mediated by additional proteins,or if other factors are involved, as the plant heteromeric ACCase hasnever been fully characterized. A comprehensive study of ACCase proteininteractions is needed.

Therefore, there is a need to provide a better understanding of proteinstructure and regulation of ACCase to leverage the potential formanipulating flux through this committed and irreversible step for denovo FAS. There is also a need to develop a novel method to efficientlyincrease ACCase activity to consequently increase fatty acid and,ultimately, triacylglycerol production in plants and algae.

SUMMARY OF THE INVENTION

In one aspect, the present invention provides methods/systems tomodulate or alter fatty acid and protein production in plants and algae.Such methods comprise the step of altering the activity level of ACCase,the committed step for de novo fatty acid biosynthesis. The inventionmay comprise the step of altering the expression of a gene family ofnegative regulators, biotin/lipoyl attachment domain containing (BADC orBLADC) proteins, which bind to the multi-subunit ACCase found in theplastids of dicotyledon and non-graminaceous monocot plants, as well asalgae. Decreasing BADC levels may enable higher oil content in algaeand/or land plants, in either vegetative or reproductive tissuesincluding, but not limited to, leaves and seeds. In addition, increasingor overexpressing expression of one or more BADC genes in a plant oralga may enable higher protein content by reducing ACCase activity andflux through de novo FAS.

According to one embodiment of the invention, the method may comprisethe step of enhancing plastid ACCase activities in the plant or algalcell by down-regulating BADC genes through biotechnology or selectivebreeding approaches. In some embodiments, total or partial silencing maycomprises antisense, RNAi, CRISPR, TALON, nanobodies, EMS, T-DNA ortransposon-mediated gene knockout, or conventional mutagenesis/targetedbreeding. In one embodiment, such a method may further comprise an RNAicassette comprising SEQ ID NOs: 7 or 138. In some embodiments, suchapproaches may comprise total or partial silencing of one or more BADCgenes, such as genes and gene orthologs of BADC1, BADC2, and/or BADC3 orartificial genes containing essential BADC motifs. In one embodiment,the one or more BADC genes may comprise from about 70% to about 100%sequence identity to a nucleotide sequence selected from the groupconsisting of SEQ ID NOs: 2, 4, 6, 7, and 138, or a complement thereof.In another embodiment, the one or more BADC genes may encode apolypeptide comprising from about 70% to about 100% sequence identity toa poypeptide sequence selected from the group consisting of SEQ ID NOs:1, 3, 5, 8-137, and 139-143. In another embodiment, the inventionprovides a plant or part thereof produced by a method described herein,wherein the plant comprises increased seed oil content, or a seed thatproduces the plant or part thereof, wherein the seed comprises increasedseed oil content. In some embodiments, such methods may be carried outin organisms that contain orthologs to the Arabidopsis thaliana BADCgenes, such as Amborella trichopoda, Arabidopsis lyrata, Arabidopsisalpine, Arachis hypogaea, Auxenochlorella protothecoides, Brassicanapus, Brassica rapa, Camelina sativa, Capsella rubella, Cathamustinctorius, Chlamydomonas reinhardtii, Chlorella variabilis, Cicerarietinum, Citrus clementina, Citrus sinensis, Coccomyxa subellipsoideasC-169, Coffea canephora, Cucumis melo, Cucumis sativus, Elaeisguineensis, Erythranthe guttata, Eucalyptus grandis, Eutremasalsugineum, Fragaria vesca, Genlisea aurea, Glycine max, Helianthusannuus, Helicosporidium ATCC 50920, Jatropha curcas, Lotus japonicas,Medicago truncatula, Morus notabilis, Musa acuminate, Nelumbo nucifera,Nicotiana sylvestris, Nicotiana tomentosiformis, Phaseolus vulgaris,Pheonix dactylifera, Physcomitrella patens, Picea sitchensis,Polytomella parva, Populus trichocarpa, Prunus mume, Prunes persica,Pyrus×bretschneideri, Ricinus communis, Selaginella moellendorffii,Sesamum indicum, Solanum lycopersicum, Solanum tuberosum, Theobromacacao, Thlaspi arvense, Vitis vinifera, or Volvox carteri.

In another embodiment of the invention, the method may comprises thestep of reducing plastid ACCase activity by up-regulating BADC genesthrough biotechnology or selective breeding approaches in an organismthat contains an ortholog to the Arabidopsis thaliana BADC genes. In oneembodiment, such up-regulation of one or more BADC genes may comprisealtering expression of one or more BADC genes in either seed orvegetative tissue of a plant or alga, such as genes and gene orthologsof BADC1, BADC2, and/or BADC3 or artificial genes containing essentialBADC motifs. In one embodiment, the one or more BADC genes may comprisefrom about 70% to about 100% sequence identity to a nucleotide sequenceselected from the group consisting of SEQ ID NOs: 2, 4, 6, 7, and 138,or a complement thereof. In another embodiment, the one or more BADCgenes may encode a polypeptide comprising from about 70% to about 100%sequence identity to a poypeptide sequence selected from the groupconsisting of SEQ ID NOs: 1, 3, 5, 8-137, and 139-143. In anotherembodiment, the invention provides a plant or part thereof produced by amethod described herein, wherein the plant comprises increased seed oilcontent, or a seed that produces the plant or part thereof, wherein theseed comprises increased seed oil content, such as Amborella trichopoda,Arabidopsis lyrata, Arabidopsis alpine, Arachis hypogaea,Auxenochlorella protothecoides, Brassica napus, Brassica rapa, Camelinasativa, Capsella rubella, Cathamus tinctorius, Chlamydomonasreinhardtii, Chlorella variabilis, Cicer arietinum, Citrus clementina,Citrus sinensis, Coccomyxa subellipsoideas C-169, Coffea canephora,Cucumis melo, Cucumis sativus, Elaeis guineensis, Erythranthe guttata,Eucalyptus grandis, Eutrema salsugineum, Fragaria vesca, Genlisea aurea,Glycine max, Helianthus annuus, Helicosporidium ATCC 50920, Jatrophacurcas, Lotus japonicas, Medicago truncatula, Morus notabilis, Musaacuminate, Nelumbo nucifera, Nicotiana sylvestris, Nicotianatomentosiformis, Phaseolus vulgaris, Pheonix dactylifera, Physcomitrellapatens, Picea sitchensis, Polytomella parva, Populus trichocarpa, Prunusmume, Prunes persica, Pyrus×bretschneideri, Ricinus communis,Selaginella moellendorffii, Sesamum indicum, Solanum lycopersicum,Solanum tuberosum, Theobroma cacao, Thlaspi arvense, Vitis vinifera, orVolvox carteri.

Further areas of applicability of the present disclosure will becomeapparent from the description provided herein. It should be understoodthat the description and specific examples are intended for purposes ofillustration only and are not intended to limit the scope of the presentteachings.

BRIEF DESCRIPTION OF THE DRAWINGS

For a more complete understanding of the features and advantages of thepresent invention, reference is now made to the detailed description ofthe invention along with the accompanying figures. The drawingsdescribed herein are for illustration purposes only and are not intendedto limit the scope of the present teachings in any way.

FIG. 1—Shows co-immunoprecipitation of ACCase and BADC proteins fromArabidopsis thaliana seedlings. (A and B) Proteins were precipitatedfrom Arabidopsis thaliana crude chloroplast lysate using antibodiesspecific for ACCase subunits BCCP2 (A) or α-CT (B) and identified byLC-MS/MS. Control precipitations were performed with uncoated Protein ASepharose beads. For both sets of studies, n=7. Error bars representstandard deviation. Semi-quantitative normalized relative abundancevalues were determined by dividing total spectral matches for eachprotein by protein size and normalizing to the antibody-specificprotein. (C and D) Protein blot analysis of reciprocal co-IPs from thesame Arabidopsis lysate showed that the BCCP subunits of ACCaseco-precipitated with BADC1 (C) and BADC2 (D). Blots are representativeof three biological replicates.

FIG. 2—Shows primer design for yeast two-hybrid and recombinantexpression cloning studies. (A) Primers used to amplify the genes shownwere ordered from Sigma-Aldrich and include: BCCP1 (accession no.AT5G16390) sense and antisense primers (SEQ ID NOs: 144 and 145,respectively); BCCP2 (accession no. AT5G15530) sense and antisenseprimers (SEQ ID NOs: 146 and 147, respectively); BADC1 (accession no.AT3G56130) sense and antisense primers (SEQ ID NOs: 148 and 149,respectively); BADC2 (accession no. AT1G52670) sense and antisenseprimers (SEQ ID NOs: 150 and 151, respectively); BADC3 (accession no.AT3G15690) sense and antisense primers (SEQ ID NOs: 152 and 153,respectively), and apo-BCCP1 (accession no. AT5G16390) mutant sense andantisense primers (SEQ ID NOs: 160 and 161, respectively). Underlinedsegments indicate a restriction endonuclease site. (B) Transit peptidelengths were predicted using TargetP. Primers were designed to omit thebases coding for these peptides from the gene of interest to allow forproper protein folding.

FIG. 3—Shows the direct interaction of BADC proteins with BCCP subunitsof ACCase. (A) Strain AH109 yeast was transformed with bait and preyconstructs containing the genes shown. Negative controls showed minimalor no growth. Sv40 and p53 were used as positive controls. Lamin C wasused as a negative control. Transformed yeast were plated on medialacking Trp, Leu, and His. Results shown are representative of threebiological replicates. (B) Coomassie-stained gels showing the elutionfractions of Ni²⁺-NTA-purified protein from E. coli. At right, a nativeprotein was co-expressed with a His₆-tagged protein. At left, the nativeprotein was expressed alone. The native proteins were present stronglyin the elution fractions only when co-expressed with the His₆-taggedprotein. Protein identities were confirmed by LC-MS/MS.

FIG. 4—Shows that BADC proteins share substantial sequence identity withBCCP subunits of ACCase in Arabidopsis thaliana. Protein sequences fromArabidopsis thaliana were aligned and percent amino acid identity wascalculated using Clustal-W.

FIG. 5—Shows predicted structures of BADC proteins resemble BCCPsubunits of ACCase in Arabidopsis thaliana. Structures of each proteinwere generated using SWISS-MODEL. Protein sequences lacking thepredicted transit peptide residues were used as the input.

FIG. 6A Shows an alignment of the C-termini of the Arabidopsis thalianaBCCP and BADC proteins demonstrating that the BADC proteins resembleBCCP isoforms but lack the conserved biotinylation motif and biotinyllysine residue. BCCP1 (SEQ ID NO:139), BCCP2 (SEQ ID NO:140), BADC1 (SEQID NO:141), BADC2 (SEQ ID NO:142), and BADC3 (SEQ ID NO:143).

FIG. 6B and FIG. 6C—Show differences in biotinylation between the BCCPand BADC proteins, illustrating that the BADC proteins resemble BCCPisoforms but are not biotinylated. (B) Western blotting analysis ofrecombinant Arabidopsis proteins using a biotin-specific antibody. BCCP2was observed to be biotinylated while the BADCs were not. (C) Proteinblot analysis of immunoprecipitated in vivo BADC1 from Arabidopsisseedlings. Blotting precipitate with BADC1-specific antibody showed thepresence BADC1 in the sample, while blotting with biotin-specificantibody showed no recognition of BADC1.

FIG. 7—Shows species containing orthologs of the Arabidopsis thalianaBADC proteins. Phylogenetic trees showing all species that wereidentified to contain an ortholog to one or more of the BADC proteins inA. thaliana. Ortholog candidates were identified by performing PSI-BLASTon the primary sequence of each BADC protein. Results were filtered byperforming a reciprocal BLAST against the A. thaliana proteome. Speciesclassification is indicated by color. All species identified containheteromeric ACCase.

FIG. 8—Shows BADC3 reduces ACCase activity in E. coli. (A) Growth curvesshowing the optical density of L8 E. coli cells over time. Cultures weregrown in M63 liquid culture at 37° C. in the absence of fatty acids.Transformed cells contained the following vectors: empty pET28a (EV28a),pET28a containing the E. coli BCCP gene (EcBCCP), empty pET11a (EV11a),and/or pET11a containing the A. thaliana BADC3 gene (BADC3). The EVcontrol cells show minimal growth at 37° C., while re-introduction ofnative EcBCCP complemented the temperature-sensitive phenotype. BADC3expression alone showed no statistical difference from EV control exceptat T=10 h, while co-expression of BADC3 with EcBCCP showed anapproximate 75% reduction in growth compared to EcBCCP alone acrossmultiple studies. Results shown are representative of three separatestudies. Error bars represent standard deviation. (B) Coomassie bluestain and anti-biotin protein blot of Ni²⁺-NTA purified His₆-BADC3.

FIG. 9—BADCs reduce ACCase activity in A. thaliana. Protein extractedfrom 10-d-old A. thaliana siliques was assayed for ACCase activity byincorporation of radiolabelled sodium bicarbonate into acid-stableproducts. Assays were performed in the absence (WT) or presence of 10 μMrecombinant BADC1, BADC2, BADC3, BCCP2, or BSA. Specific activities werecalculated for each assay and then normalized to WT control. Fourbiological replicates were performed for each trial. Error bars denoteSEM.

FIG. 10—Shows light-dependent changes in gene expression of BADC andhetACCase in A. thaliana siliques. (A) Graph shows the absoluteexpression level of the given genes obtained by qPCR. Ten d old A.thaliana siliques were collected after various amounts of lightexposure. RNA extracted from these tissues was used to create cDNA forthis analysis. Average values of four biological replicates are shown.Standard error was approximately 5 to 10 percent for all data points.(B) Graph depicts the shift in BADC and BCCP total transcript level inresponse to light. The sum of transcript levels from BCCPs and BADCs in(A) for each time point were normalized to the sum of BCCP transcriptsat six hours light exposure. At T=0, the ratio of BADC:BCCP transcriptis 9:1. At T=6, the ratio shifts to 1:4.

FIG. 11—Shows a bar graph illustrating seed oil content of 26 mutantBADC1 RNAi lines and one wild type. Statistical significance wasdetermined by Student's t-test (*, P<0.05).

FIG. 12—Seed specific RNAi silencing of BADC1 increases seed oil contentin A. thaliana. (A) Bar graph shows total seed oil content in WT andbasta-resistant T2 A. thaliana lines containing a construct thatsilences BADC1 expression in the seed. Each bar represents the averageof four plants. Error bars denote SD. (B) RT-PCR analysis of BADC1 RNAisilencing lines. BADC1 transcript level was quantified relative to Actintranscript level and normalized to WT. RNA used for analysis wasextracted from four biological replicates of ten d old siliques. Errorbars denote SEM. In both graphs, statistical significance was determinedby Student's t-test (*, P<0.05).

FIG. 13A—Shows a scatter plot demonstrating the expression profiles ofACCase and BADC genes in developing seed of Arabidopsis thaliana usingpublicly available transcriptomics data.

FIG. 13B—Shows a scatter plot demonstrating the expression profiles ofACCase and BADC genes in developing seed of Glycine max.

FIG. 14—Shows dose-dependent inhibition of ACCase activity by BADC1. (A)Shows ACCase activity from 20-day-old A. thaliana leaf extracts, averageof four biological replicates. Error bars denote standard deviation. (B)Shows a table of the values from (A). (C) shows SDS-PAGE analysis ofpurified recombinant BADC1 protein used in assays for panel (A).

FIG. 15—Shows a model of negative regulation of ACCase by BADC. TheBC-BCCP subcomplex of ACCase consists of two dimers of BC and four BCCPproteins. A model was proposed in which BADC proteins compete with BCCPfor binding to BC. Binding of BADC prevents binding of the essentialBCCP subunit. The pool of BC/BCCP and BC/BCCP/BADC subcomplexes thencompete for interaction with the CT subcomplex, leading to variablereductions in ACCase activity. While a transient association of the twoACCase half reactions is known, it is unclear whether BADC can displaceBCCP from an assembled BC/BCCP subcomplex (dashed arrows).Abbreviations: BC, biotin carboxylase; BCCP, biotin carboxyl carrierprotein; BADC, biotin/lipoyl attachment domain protein; CT,carboxyltransferase.

BRIEF DESCRIPTION OF THE SEQUENCES

SEQ ID NO: 1—BADC1 polypeptide sequence, AT3G56130, biotin/lipoylattachment domain-containing protein.

SEQ ID NO: 2—BADC1 full genomic nucleic acid sequence, AT3G56130,biotin/lipoyl attachment domain-containing protein.

SEQ ID NO: 3—BADC2 polypeptide, AT1G52670, Single hybrid motif protein.

SEQ ID NO: 4—BADC2 full genomic nucleic acid, AT1G52670, Single hybridmotif protein.

SEQ ID NO: 5—BADC3 polypeptide, AT3G15690, Single hybrid motif protein.

SEQ ID NO: 6—BADC3 full genomic nucleic acid, AT3G15690, Single hybridmotif protein.

SEQ ID NO: 7—Nucleic acid, RNAi cassette for BADC1 silencing inArabidopsis thaliana.

SEQ ID NOs: 8-134—Polypeptide sequences of each BADC ortholog acrossvarious organisms. Orthologous proteins were identified by performing aPSI-BLAST search using the protein sequence of each BADC fromArabidopsis thaliana as the query against known plant and algaeproteomes. Orthologs were confirmed by reciprocal BLAST search againstthe Arabidopsis thaliana proteome. Sequences show GenBank ID, referencenumber, protein annotation, and name of the genus and species.

SEQ ID NOs: 135-137—Show the consensus sequence identified by multiplesequence alignment of all identified BADC orthologs and the three BADCisoforms in Arabidopsis thaliana. This consensus sequence identifies aprotein as a BADC ortholog. SEQ ID NO: 135—Polypeptide sequence ofinternal 44 amino acids of Arabidopsis thaliana BADC1 protein, accessionAT3G56130. The 44 amino acids are conserved among the BADC proteinfamily.

SEQ ID: 136—Internal 44 amino acids of BADC consensus motif 1, withidentical amino acid residues at positions 1, 2, 11, 12, 28, 29, 36, 38,and 42.

SEQ ID NO: 137—Internal 44 amino acids of BADC consensus motif 1, withvariable amino acid residues at positions 1, 2, 11, 12, 28, 29, 36, 38,and 42, and providing variable residues.

SEQ ID NO: 138—Nucleic acid, RNAi cassette to target BADC1 and BADC3genes in Glycine max.

SEQ ID NO: 139—BCCP1 protein sequence.

SEQ ID NO: 140—BCCP2 protein sequence.

SEQ ID NO: 141—BADC1 protein sequence.

SEQ ID NO: 142—BADC2 protein sequence.

SEQ ID NO: 143—BADC3 protein sequence.

SEQ ID NOs: 144 and 145—Sequences of BCCP1 (accession no. AT5G16390)sense and antisense primers, respectively.

SEQ ID NOs: 146 and 147—Sequences of BCCP2 (accession no. AT5G15530)sense and antisense primers, respectively.

SEQ ID NOs: 148 and 149—Sequences of BADC1 (accession no. AT3G56130)sense and antisense primers, respectively.

SEQ ID NOs: 150 and 151—Sequences of BADC2 (accession no. AT1G52670)sense and antisense primers, respectively.

SEQ ID NOs: 152 and 153—Sequences of BADC3 (accession no. AT3G15690)sense and antisense primers, respectively.

SEQ ID NO: 154 and 155—Primer Sequences used to amplify AtBADC1.

SEQ ID NOs: 156—Sequence of sense primer used to amplify BADC1.

SEQ ID NOs: 157—Sequence of antisense primer used to amplify BADC1.

SEQ ID NOs: 158—Sequence of sense primer used to amplify Actin 8.

SEQ ID NOs: 159—Sequence of antisense primer used to amplify Actin 8.

SEQ ID NOs: 160 and 161—Sequences of apo-BCCP1 (accession no. AT5G16390)mutant sense and antisense primers, respectively.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides methods and means to modulate fatty acid,and ultimately triacylglycerol, production, as well as proteinproduction, in plants and algae. Such methods comprise altering theactivity levels of the committed step for de novo fatty acidbiosynthesis, catalyzed by acetyl-CoA carboxylases (ACCase). Inaccordance with the invention, a method described herein may increase ordecrease ACCase activity levels by down- or up-regulating thebiotin/lipoyl attachment domain containing (BADC) genes, respectively.

The BADC proteins are a family of three proteins in Arabidopsis thalianaand resemble the biotin carboxyl carrier protein (BCCP) subunit toACCase. BADC protein expression has a negative effect on ACCaseactivity, which in turn affects oil production in plants and algae. Theactivity of ACCase in catalyzing the committed step of de novo fattyacid synthesis and regulation of flux through this central metabolicpathway is known in the art. In dicot and non-graminaceous monocotplants and algae, plastid ACCase is a heteromeric complex comprised offour catalytic subunits: biotin carboxylase (BC), biotin carboxylcarrier protein (BCCP), and α- and β-carboxyltransferase (α-CT, β-CT).The plant complex is recalcitrant to conventional purification schemesand hence the structure and composition of the full assembly is unknown.

As described in detail below, in vivo co-immunoprecipitation withsubunit-specific antibodies was used to identify a novel family of BADCproteins, provided herein as SEQ ID NOs: 1-6, in Arabidopsis thaliana.It was determined that BADC proteins resemble BCCP (subunits of ACCasecomplex) but cannot be biotinylated, and based on the results fromorthogonal techniques, all three BADC proteins interact with the two A.thaliana BCCP isoforms and the biotin carboxylase subunit of ACCase,based on yeast two-hybrid and heterologous co-expression analyses. Noneof the BADC proteins were biotinylated in planta or when expressed inEscherichia coli, unlike BCCP controls. Gene orthologs to BADC werefound only in plant and green algae species that contain a heteromericACCase suggesting BADC genes co-evolved with this form of ACCase. It wasfurther discovered that expression of BADC proteins inhibited ACCaseactivity when co-expressed with a functional BCCP in atemperature-sensitive Eschericia coli BCCP mutant. Thus, BADC proteinsregulate ACCase by competing with BCCP to form less active complexes.Down-regulating BADC genes (i.e., silencing the expression of BADCprotein) promotes the formation of active ACCase complexes, which inturn increases ACCase activity levels and thus oil production in plantsand/or algae. Down-regulating one or more BADC genes may be achieved viavarious biotechnology or selective breeding approaches as describedherein and/or known in the art.

The invention also provides methods of reducing fatty acid synthesis inplants and/or algae by overexpression of BADC genes. Such methods wouldreduce the amount of triacylglycerol stored in the seed andconcomitantly increase the amount of protein stored in the seed.Overexpression of BADC genes may therefore reduce ACCase activity and inturn reduce fatty acid biosynthesis.

The present disclosure provides a method of marker-assisted selection asa screening tool for plant and/or algae species that contain higher oilcontent. The BADC genes are traits that can be monitored to select forspecific organisms that may have the potential to produce moretriacylglycerol. The expression level of BADC genes may be the markerused to assist in such selection, wherein organisms with naturallyreduced expression of BADC genes may be selected.

Plant oils are an important renewable source of hydrocarbons for food,energy, and industrial feedstocks. Acyl chains stored as triacylglycerolare produced by the de novo fatty acid synthesis (FAS) pathway. Thecommitted step of de novo FAS is catalyzed by the heteromericacetyl-coenzyme A carboxylase (hetACCase) which carboxylates acetyl-CoAto form malonyl-CoA in a two-step reaction requiring ATP, bicarbonate,and biotin cofactor. In prokaryotes, and in plastids of dicots andnon-graminaceous monocots, hetACCase is a heteromeric complex requiringfour distinct subunits: biotin carboxylase (BC), biotin carboxyl carrierprotein (BCCP), and α- and β-carboxyltransferase (CT). Graminaceousmonocots possess a homomeric form of plastid ACCase wherein thecatalytic components are fused in tandem as a single polypeptide.Structural models for hetACCase are based on studies of the Escherichiacoli homolog. The E. coli hetACCase is composed of two enzymaticsubcomplexes: an α/β-CT heterotetramer and a BC/BCCP heterooctamer. Thecomponents of each subcomplex form stable associations while the twosubcomplexes themselves show a relatively weak interaction with oneanother. This property has contributed to the difficulties inbiochemical and structural characterization of hetACCase from plants.

Without being limited to a particular theory, a plant useful for thepresent invention may be include, but is not limited to, plant or algalapecies, such as Amborella trichopoda, Arabidopsis lyrata, Arabidopsisalpine, Auxenochlorella protothecoides, Brassica napus, Brassica rapa,Capsella rubella, Chlamydomonas reinhardtii, Chlorella variabilis, Cicerarietinum, Citrus clementina, Citrus sinensis, Coccomyxa subellipsoideasC-169, Coffea canephora, Cucumis melo, Cucumis sativus, Elaeisguineensis, Erythranthe guttata, Eucalyptus grandis, Eutremasalsugineum, Fragaria vesca, Genlisea aurea, Glycine max, Helianthusannuus, Helicosporidium ATCC 50920, Jatropha curcas, Lotus japonicas,Medicago truncatula, Morus notabilis, Musa acuminate, Nelumbo nucifera,Nicotiana sylvestris, Nicotiana tomentosiformis, Phaseolus vulgaris,Pheonix dactylifera, Physcomitrella patens, Picea sitchensis,Polytomella parva, Populus trichocarpa, Prunus mume, Prunes persica,Pyrus×bretschneideri, Ricinus communis, Selaginella moellendorffii,Solanum lycopersicum, Solanum tuberosum, Theobroma cacao, Vitisvinifera, or Volvox carteri.

I. Nucleic Acids, Polypeptides and Plant Transformation Constructs

Certain embodiments of the current invention concern isolated nucleicacid sequences and the corresponding polypeptode sequences for a novelfamily of BADC proteins, provided herein as SEQ ID NOs:1-6, inArabidopsis thaliana. Complements to any nucleic acid or proteinsequences described herein are also provided.

“Identity,” as is well understood in the art, is a relationship betweentwo or more polypeptide sequences or two or more polynucleotidesequences, as determined by comparing the sequences. In the art,“identity” also means the degree of sequence relatedness betweenpolypeptide or polynucleotide sequences, as determined by the matchbetween strings of such sequences. Methods to determine “identity” aredesigned to give the largest match between the sequences tested.Moreover, methods to determine identity are codified in publiclyavailable programs. “Identity” can be readily calculated by knownmethods including, but not limited to, those described in Lesk, ed.,(1988); Smith, ed., (1993); Griffin, and Griffin, eds., (1994); vonHeinje, (1987); Gribskov and Devereux, eds., (1991); and Carillo andLipman, (1988). Computer programs can be used to determine “identity”between two sequences these programs include but are not limited to, GCG(Devereux, 1984); suite of five BLAST programs, three designed fornucleotide sequences queries (BLASTN, BLASTX, and TBLASTX) and twodesigned for protein sequence queries (BLASTP and TBLASTN) (Coulson,1994; Birren, et al., 1997). The BLASTX program is publicly availablefrom NCBI and other sources (BLAST Manual, Altschul, S., et al., NCBINLM NIH, Bethesda, Md. 20894; Altschul, S., et al., 1990). The wellknown Smith Waterman algorithm can also be used to determine identity.

In accordance with the invention, a polynucleotide or polypeptidesequence as described herein may exhibit at least from about 70% toabout 100% sequence identity to at least one of the sequences set forthherein. For example, in one embodiment, a BADC gene as described hereinmay comprise, for example, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%,79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%,93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to asequence selected from SEQ ID NO:2, 4, 6, 7, or 138, or a complementthereof. In other embodiments, a BADC protein as described herein maycomprise for example, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%,80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%,94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to a sequenceselected from SEQ ID NO:1, 3, or 5, or a complement thereof.

Parameters for polypeptide sequence comparison include the following:Algorithm: Needleman and Wunsch (1970); Comparison matrix: BLOSUM62 fromHentikoff and Hentikoff, (1992); Gap Penalty: 12; and Gap LengthPenalty: 4. A program which can be used with these parameters ispublicly available as the “gap” program from Genetics Computer Group,Madison Wis. The above parameters along with no penalty for end gap mayserve as default parameters for peptide comparisons.

Parameters for nucleic acid sequence comparison include the following:Algorithm: Needleman and Wunsch (1970); Comparison matrix: matches=+10;mismatches=0; Gap Penalty: 50; and Gap Length Penalty: 3. A programwhich can be used with these parameters is publicly available as the“gap” program from Genetics Computer Group, Madison Wis. The aboveparameters may serve as the default parameters for nucleic acidcomparisons.

As used herein, “hybridization,” “hybridizes,” or “capable ofhybridizing” is understood to mean the forming of a double- ortriple-stranded molecule or a molecule with partial double- ortriple-stranded nature. Such hybridization may take place underrelatively high-stringency conditions, including low salt and/or hightemperature conditions, such as provided by a wash in about 0.02 M toabout 0.15 M NaCl at temperatures of about 50° C. to about 70° C. for 10min. In one embodiment of the invention, the conditions are 0.15 M NaCland 70° C. Stringent conditions tolerate little mismatch between anucleic acid and a target strand. Such conditions are well known tothose of ordinary skill in the art, and are preferred for applicationsrequiring high selectivity. Non-limiting applications include isolatinga nucleic acid, such as a gene or a nucleic acid segment thereof, ordetecting at least one specific mRNA transcript or a nucleic acidsegment thereof, and the like. Also included may be a protein orpolypeptide, or fragment thereof, such as any of those set forth herein.

The nucleic acids provided herein as SEQ ID NOs:1-6 may be from anysource, e.g., identified as naturally occurring in a plant, orsynthesized, e.g., by mutagenesis of SEQ ID NOs:1-6. In an embodiment,the naturally occurring sequence may be from any plant or algal species,such as Amborella trichopoda, Arabidopsis lyrata, Arabidopsis alpine,Auxenochlorella protothecoides, Brassica napus, Brassica rapa, Capsellarubella, Chlamydomonas reinhardtii, Chlorella variabilis, Cicerarietinum, Citrus clementina, Citrus sinensis, Coccomyxa subellipsoideasC-169, Coffea canephora, Cucumis melo, Cucumis sativus, Elaeisguineensis, Erythranthe guttata, Eucalyptus grandis, Eutremasalsugineum, Fragaria vesca, Genlisea aurea, Glycine max, Helianthusannuus, Helicosporidium ATCC 50920, Jatropha curcas, Lotus japonicas,Medicago truncatula, Morus notabilis, Musa acuminate, Nelumbo nucifera,Nicotiana sylvestris, Nicotiana tomentosiformis, Phaseolus vulgaris,Pheonix dactylifera, Physcomitrella patens, Picea sitchensis,Polytomella parva, Populus trichocarpa, Prunus mume, Prunes persica,Pyrus×bretschneideri, Ricinus communis, Selaginella moellendorffii,Solanum lycopersicum, Solanum tuberosum, Theobroma cacao, Vitisvinifera, or Volvox carteri.

Coding sequences may be provided in a recombinant vector operably linkedto a heterologous promoter functional in plants, in either sense orantisense orientation. Expression constructs may also be providedcomprising these sequences, including antisense oligonucleotidesthereof. In other embodiments, plants and plant cells transformed withthe sequences may be provided. The construction of vectors which may beemployed in conjunction with plant transformation techniques using theseor other sequences according to the invention will be known to those ofskill of the art in light of the present disclosure (see, for example,Sambrook et al., 1989; Gelvin et al., 1990). The techniques of thecurrent invention are thus not limited to any particular nucleic acidsequences.

The choice of any additional elements used in conjunction with a codingsequences or corresponding encoded product may depend on the purpose ofthe transformation. One of the major purposes of transformation of cropplants is to add commercially desirable, agronomically important traitsto the plant, as described herein.

Vectors used for plant transformation may include, for example,plasmids, cosmids, YACs (yeast artificial chromosomes), BACs (bacterialartificial chromosomes) or any other suitable cloning system, as well asfragments of DNA therefrom. Thus when the term “vector” or “expressionvector” is used, all of the foregoing types of vectors, as well asnucleic acid sequences isolated therefrom, are included. It iscontemplated that utilization of cloning systems with large insertcapacities will allow introduction of large DNA sequences comprisingmore than one selected gene. In accordance with the invention, thiscould be used to introduce genes corresponding to, e.g., an entirebiosynthetic pathway, into a plant.

Particularly useful for transformation are expression cassettes whichhave been isolated from such vectors. DNA segments used for transformingplant cells will generally comprise the cDNA, gene, or genes which onedesires to introduce into and have expressed in the host cells. TheseDNA segments can further include structures such as promoters,enhancers, polylinkers, or even regulatory genes as desired. The DNAsegment or gene chosen for cellular introduction will often encode aprotein which will be expressed in the resultant recombinant cellsresulting in a screenable or selectable trait and/or which will impartan improved phenotype to the resulting transgenic plant. Preferredcomponents likely to be included with vectors used in the currentinvention are as follows.

A. Regulatory Elements

Exemplary promoters for expression of a nucleic acid sequence includeplant promoters such as the CaMV 35S promoter (Odell et al., 1985), orothers such as CaMV 19S (Lawton et al., 1987), nos (Ebert et al., 1987),Adh (Walker et al., 1987), sucrose synthase (Yang and Russell, 1990),α-tubulin, actin (Wang et al., 1992), cab (Sullivan et al., 1989),PEPCase (Hudspeth and Grula, 1989) or those promoters associated withthe R gene complex (Chandler et al., 1989). Tissue-specific promoterssuch as leaf specific promoters, or tissue selective promoters (e.g.,promoters that direct greater expression in leaf primordia than in othertissues), and tissue-specific enhancers (Fromm et al., 1986) are alsocontemplated to be useful, as are inducible promoters such as ABA- andturgor-inducible promoters. Any suitable promoters known in the art maybe used to express coding sequences in a plant.

The DNA sequence between the transcription initiation site and the startof the coding sequence, i.e., the untranslated leader sequence, can alsoinfluence gene expression. One may thus wish to employ a particularleader sequence with a transformation construct of the invention. In anembodiment, leader sequences are contemplated to include those whichcomprise sequences predicted to direct optimum expression of theattached gene, i.e., to include a consensus leader sequence which mayincrease or maintain mRNA stability and prevent inappropriate initiationof translation. The choice of such sequences will be known to those ofskill in the art in light of the present disclosure.

It is envisioned that a sequence useful for altering activity levels ofACCase as described herein may comprise any sequence set forth herein,for example SEQ ID NOs:1-6. In certain embodiments, a gene useful foraltering ACCase levels may comprise altering expression of a BADC gene,such as BADC1, BADC2, BADC3, set forth herein as SEQ ID NOs:2, 4, and 6,respectively, or orthologs or homologs thereof. Such an ortholog orhomolog may be from any species useful in accordance with the invention.Such a sequence may be introduced into a plant under the control ofnovel promoters, enhancers, etc., or homologous or tissue-specific ortissue-selective promoters or control elements. Vectors for use intissue-specific targeting of genes in transgenic plants will typicallyinclude tissue-specific or tissue-selective promoters and may alsoinclude other tissue-specific or tissue-selective control elements suchas enhancer sequences. Promoters which direct specific or enhancedexpression in certain plant tissues will be known to those of skill inthe art in light of the present disclosure. These include, for example,the rbcS promoter, specific for green tissue; the ocs, nos and maspromoters, which have higher activity in roots; or napin and glycininpromoters, which have higher activity in developing seed.

B. Terminators

Transformation constructs prepared in accordance with the invention mayinclude a 3′ end DNA sequence that acts as a signal to terminatetranscription and allow for the polyadenylation of the mRNA produced bycoding sequences operably linked to a promoter. In one embodiment of theinvention, the native terminator of a BADC coding sequence may be used.Alternatively, a heterologous 3′ end may enhance the expression of senseor antisense BADC coding sequences. Examples of terminators that may beused in this context include those from the nopaline synthase gene ofAgrobacterium tumefaciens (nos 3′ end) (Bevan et al., 1983), theterminator for the T7 transcript from the octopine synthase gene ofAgrobacterium tumefaciens, and the 3′ end of the protease inhibitor I orII gene from potato or tomato. Regulatory elements such as an Adh intron(Callis et al., 1987), sucrose synthase intron (Vasil et al., 1989) orTMV omega element (Gallie et al., 1989), may further be included wheredesired.

C. Transit or Signal Peptides

Sequences that are joined to the coding sequence of an expressed gene,which are removed post-translationally from the initial translationproduct and which facilitate the transport of the protein into orthrough intracellular or extracellular membranes, are termed transit(usually into vacuoles, vesicles, plastids and other intracellularorganelles) and signal sequences (usually to the endoplasmic reticulum,Golgi apparatus, and outside of the cellular membrane). By facilitatingthe transport of the protein into compartments inside and outside thecell, these sequences may increase the accumulation of gene products byprotecting them from proteolytic degradation. These sequences also allowfor additional mRNA sequences from highly expressed genes to be attachedto the coding sequence of the genes. Since mRNA being translated byribosomes is more stable than naked mRNA, the presence of translatablemRNA in front of the gene may increase the overall stability of the mRNAtranscript from the gene and thereby increase synthesis of the geneproduct. Since transit and signal sequences are usuallypost-translationally removed from the initial translation product, theuse of these sequences allows for the addition of extra translatedsequences that may not appear on the final polypeptide. It further iscontemplated that targeting of certain proteins may be desirable inorder to enhance the stability of the protein (U.S. Pat. No. 5,545,818,incorporated herein by reference in its entirety).

Additionally, vectors may be constructed and employed in theintracellular targeting of a specific gene product within the cells of atransgenic plant or in directing a protein to the extracellularenvironment. This generally will be achieved by joining a DNA sequenceencoding a transit or signal peptide sequence to the coding sequence ofa particular gene. The resultant transit or signal peptide willtransport the protein to a particular intracellular or extracellulardestination, respectively, and will then be post-translationallyremoved.

D. Marker Genes

By employing a selectable or screenable marker, one can provide orenhance the ability to identify transformants. “Marker genes” are genesthat impart a distinct phenotype to cells expressing the marker proteinand thus allow such transformed cells to be distinguished from cellsthat do not have the marker. Such genes may encode either a selectableor screenable marker, depending on whether the marker confers a traitwhich one can “select” for by chemical means, i.e., through the use of aselective agent (e.g., a herbicide, antibiotic, or the like), or whetherit is simply a trait that one can identify through observation ortesting, i.e., by “screening” (e.g., the green fluorescent protein). Ofcourse, many examples of suitable marker proteins are known to the artand can be employed in the practice of the invention.

Many selectable marker coding regions are known and could be used withthe present invention including, but not limited to, neo (Potrykus etal., 1985), which provides kanamycin resistance and can be selected forusing kanamycin, G418, paromomycin, etc.; bar, which confers bialaphosor phosphinothricin resistance; a mutant EPSP synthase protein (Hincheeet al., 1988) conferring glyphosate resistance; a nitrilase such as bxnfrom Klebsiella ozaenae which confers resistance to bromoxynil (Stalkeret al., 1988); a mutant acetolactate synthase (ALS) which confersresistance to imidazolinone, sulfonylurea or other ALS inhibitingchemicals (European Patent Application 154, 204, 1985); a methotrexateresistant DHFR (Thillet et al., 1988), a dalapon dehalogenase thatconfers resistance to the herbicide dalapon; or a mutated anthranilatesynthase that confers resistance to 5-methyl tryptophan.

An illustrative embodiment of selectable marker capable of being used insystems to select transformants are those that encode the enzymephosphinothricin acetyltransferase, such as the bar gene fromStreptomyces hygroscopicus or the pat gene from Streptomycesviridochromogenes. The enzyme phosphinothricin acetyl transferase (PAT)inactivates the active ingredient in the herbicide bialaphos,phosphinothricin (PPT). PPT inhibits glutamine synthetase, (Murakami etal., 1986; Twell et al., 1989) causing rapid accumulation of ammonia andcell death.

One beneficial use of the sequences provided by the invention may be inthe alteration of plant phenotypes such as fatty acid or triacylglycerolproduction, as well as protein production, in plants and/or algae bygenetic transformation with a coding sequence set forth herein, such asa BADC coding sequence. A BADC coding sequence such as described hereinmay be provided with other sequences. Where an expressible coding regionthat is not necessarily a marker coding region is employed incombination with a marker coding region, one may employ the separatecoding regions on either the same or different DNA segments fortransformation. In the latter case, the different vectors are deliveredconcurrently to recipient cells to maximize cotransformation.

II. Genetic Transformation

Additionally provided herein are transgenic plants transformed with theabove-identified recombinant vector encoding a BADC, or a sequencemodulating expression thereof.

Suitable methods for transformation of plant or other cells for use withthe current invention are believed to include virtually any method bywhich DNA can be introduced into a cell, such as by direct delivery ofDNA such as by PEG-mediated transformation of protoplasts (Omirulleh etal., 1993), by desiccation/inhibition-mediated DNA uptake (Potrykus etal., 1985), by electroporation (U.S. Pat. No. 5,384,253, specificallyincorporated herein by reference in its entirety), by agitation withsilicon carbide fibers (Kaeppler et al., 1990; U.S. Pat. No. 5,302,523,specifically incorporated herein by reference in its entirety; and U.S.Pat. No. 5,464,765, specifically incorporated herein by reference in itsentirety), by Agrobacterium-mediated transformation (U.S. Pat. Nos.5,591,616 and 5,563,055; both specifically incorporated herein byreference) and by acceleration of DNA coated particles (U.S. Pat. Nos.5,550,318; 5,538,877; and 5,538,880; each specifically incorporatedherein by reference in its entirety), etc. Through the application oftechniques such as these, the cells of virtually any plant species maybe stably transformed, and these cells developed into transgenic plants.

Agrobacterium-mediated transfer is a widely applicable system forintroducing genes into plant cells because the DNA can be introducedinto whole plant tissues, thereby bypassing the need for regeneration ofan intact plant from a protoplast. The use of Agrobacterium-mediatedplant integrating vectors to introduce DNA into plant cells is wellknown in the art. See, for example, the methods described by Fraley etal., (1985), Rogers et al., (1987) and U.S. Pat. No. 5,563,055,specifically incorporated herein by reference in its entirety.

Agrobacterium-mediated transformation is most efficient indicotyledonous plants and is the preferable method for transformation ofdicots, including Arabidopsis, tobacco, tomato, alfalfa and potato.Indeed, while Agrobacterium-mediated transformation has been routinelyused with dicotyledonous plants for a number of years, including alfalfa(Thomas et al., 1990), it has only recently become applicable tomonocotyledonous plants. Advances in Agrobacterium-mediatedtransformation techniques have now made the technique applicable tonearly all monocotyledonous plants. For example, Agrobacterium-mediatedtransformation techniques have now been applied to rice (Hiei et al.,1997; U.S. Pat. No. 5,591,616, specifically incorporated herein byreference in its entirety), wheat (McCormac et al., 1998), barley(Tingay et al., 1997; McCormac et al., 1998) and maize (Ishidia et al.,1996).

Modern Agrobacterium transformation vectors are capable of replicationin E. coli as well as Agrobacterium, allowing for convenientmanipulations as described (Klee et al., 1985). Moreover, recenttechnological advances in vectors for Agrobacterium-mediated genetransfer have improved the arrangement of genes and restriction sites inthe vectors to facilitate the construction of vectors capable ofexpressing various polypeptide coding genes. The vectors described(Rogers et al., 1987) have convenient multi-linker regions flanked by apromoter and a polyadenylation site for direct expression of insertedpolypeptide coding genes and are suitable for present purposes. Gateway™and other recombination-based cloning technology is also available invectors useful for plant transformation. In addition, Agrobacteriumcontaining both armed and disarmed Ti genes can be used for thetransformations. In those plant strains where Agrobacterium-mediatedtransformation is efficient, it is the method of choice because of thefacile and defined nature of the gene transfer.

One also may employ protoplasts for electroporation transformation ofplants (Bates, 1994; Lazzeri, 1995). For example, the generation oftransgenic soybean plants by electroporation of cotyledon-derivedprotoplasts is described by Dhir and Widholm in Intl. Patent Appl. Publ.No. WO 9217598 (specifically incorporated herein by reference). Otherexamples of species for which protoplast transformation has beendescribed include barley (Lazerri, 1995), sorghum (Battraw et al.,1991), maize (Bhattacharjee et al., 1997), wheat (He et al., 1994) andtomato (Tsukada, 1989).

Another method for delivering transforming DNA segments to plant cellsin accordance with the invention is microprojectile bombardment (U.S.Pat. Nos. 5,550,318; 5,538,880; 5,610,042; and PCT Application WO94/09699; each of which is specifically incorporated herein by referencein its entirety). In this method, particles may be coated with nucleicacids and delivered into cells by a propelling force. Exemplaryparticles include those comprised of tungsten, platinum, and preferably,gold. It is contemplated that in some instances DNA precipitation ontometal particles would not be necessary for DNA delivery to a recipientcell using microprojectile bombardment. However, it is contemplated thatparticles may contain DNA rather than be coated with DNA. Hence, it isproposed that DNA-coated particles may increase the level of DNAdelivery via particle bombardment but are not, in and of themselves,necessary.

An illustrative embodiment of a method for delivering DNA into plantcells by acceleration is the Biolistics Particle Delivery System, whichcan be used to propel particles coated with DNA or cells through ascreen, such as a stainless steel or Nytex screen, onto a filter surfacecovered with monocot plant cells cultured in suspension. The screendisperses the particles so that they are not delivered to the recipientcells in large aggregates. Microprojectile bombardment techniques arewidely applicable, and may be used to transform virtually any plantspecies. Examples of species for which have been transformed bymicroprojectile bombardment include monocot species such as maize (PCTApplication WO 95/06128), barley (Ritala et al., 1994; Hensgens et al.,1993), wheat (U.S. Pat. No. 5,563,055, specifically incorporated hereinby reference in its entirety), rice (Hensgens et al., 1993), oat (Torbetet al., 1995; Torbet et al., 1998), rye (Hensgens et al., 1993),sugarcane (Bower et al., 1992), and sorghum (Casa et al., 1993; Hagio etal., 1991); as well as a number of dicots including tobacco (Tomes etal., 1990; Buising and Benbow, 1994), soybean (U.S. Pat. No. 5,322,783,specifically incorporated herein by reference in its entirety),sunflower (Knittel et al. 1994), peanut (Singsit et al., 1997), cotton(McCabe and Martinell, 1993), tomato (VanEck et al. 1995), and legumesin general (U.S. Pat. No. 5,563,055, specifically incorporated herein byreference in its entirety).

The transgenic plants of the present invention expressing heterologousBADC can be of any plant or algal apecies, such as Amborella trichopoda,Arabidopsis lyrata, Arabidopsis alpine, Auxenochlorella protothecoides,Brassica napus, Brassica rapa, Capsella rubella, Chlamydomonasreinhardtii, Chlorella variabilis, Cicer arietinum, Citrus clementina,Citrus sinensis, Coccomyxa subellipsoideas C-169, Coffea canephora,Cucumis melo, Cucumis sativus, Elaeis guineensis, Erythranthe guttata,Eucalyptus grandis, Eutrema salsugineum, Fragaria vesca, Genlisea aurea,Glycine max, Helianthus annuus, Helicosporidium ATCC 50920, Jatrophacurcas, Lotus japonicas, Medicago truncatula, Morus notabilis, Musaacuminate, Nelumbo nucifera, Nicotiana sylvestris, Nicotianatomentosiformis, Phaseolus vulgaris, Pheonix dactylifera, Physcomitrellapatens, Picea sitchensis, Polytomella parva, Populus trichocarpa, Prunusmume, Prunes persica, Pyrus×bretschneideri, Ricinus communis,Selaginella moellendorffii, Solanum lycopersicum, Solanum tuberosum,Theobroma cacao, Vitis vinifera, or Volvox carteri. The plant can be anR₀ transgenic plant (i.e., a plant derived from the original transformedtissue). The plant can also be a progeny plant of any generation of anR₀ transgenic plant, wherein the transgenic plant has the nucleic acidsequence from the R₀ transgenic plant.

Seeds of the any above-described transgenic plants may also be provided,particularly where the seed comprises the nucleic acid sequence.Additionally contemplated are host cells transformed with theabove-identified recombinant vector. In some embodiments, the host cellis a plant cell.

Also contemplated herein is a plant genetically engineered to increaseexpression of a BADC, where the BADC comprises a protein product of SEQID NOs: 2, 4, 6, 7, or 138, where the protein product (e.g. apolypeptide) alters plant morphology as described herein. Such a proteinproduct may comprise SEQ ID NOs:1, 3, or 5, or any other sequencedescribed herein that is appropriate for use with the present invention.In an embodiment, the altered plant morphology may be increased ordecreased fatty acid content. Such altered morphology may beaccomplished by increasing or decreasing ACCase activity levels by down-or up-regulating a BADC gene described herein. Such plants are describedin the Examples, and may be useful, e.g., as commercial plants.

The plants of these embodiments having altered expression of ACCase orone or more BADC genes may be of any species. The species may be anymonocotyledonous or dicotyledonous plant, such as those describedherein. One of skill in the art will recognize that the presentinvention may be applied to plants of other species by employing methodsdescribed herein and others known in the art.

Application of these systems to different plant strains depends upon theability to regenerate that particular plant strain from protoplasts.Illustrative methods for the regeneration of cereals from protoplastshave been described (Toriyama et al., 1986; Yamada et al., 1986;Abdullah et al., 1986; Omirulleh et al., 1993 and U.S. Pat. No.5,508,184; each specifically incorporated herein by reference in itsentirety). Examples of the use of direct uptake transformation of cerealprotoplasts include transformation of rice (Ghosh-Biswas et al., 1994),sorghum (Battraw and Hall, 1991), barley (Lazerri, 1995), oat (Zheng andEdwards, 1990) and maize (Omirulleh et al., 1993).

Tissue cultures may be used in certain transformation techniques for thepreparation of cells for transformation and for the regeneration ofplants therefrom. Maintenance of tissue cultures requires use of mediaand controlled environments. “Media” refers to the numerous nutrientmixtures that are used to grow cells in vitro, that is, outside of theintact living organism. A medium usually is a suspension of variouscategories of ingredients (salts, amino acids, growth regulators,sugars, buffers) that are required for growth of most cell types.However, each specific cell type requires a specific range of ingredientproportions for growth, and an even more specific range of formulas foroptimum growth. The rate of cell growth also will vary among culturesinitiated with the array of media that permit growth of that cell type.

Tissue that can be grown in a culture includes meristem cells, Type I,Type II, and Type III callus, immature embryos and gametic cells such asmicrospores, pollen, sperm, and egg cells. Type I, Type II, and Type IIIcallus may be initiated from tissue sources including, but not limitedto, immature embryos, seedling apical meristems, root, leaf, microsporesand the like. Those cells which are capable of proliferating as callusalso are recipient cells for genetic transformation.

Somatic cells are of various types. Embryogenic cells are one example ofsomatic cells which may be induced to regenerate a plant through embryoformation. Non-embryogenic cells are those which typically will notrespond in such a fashion. Certain techniques may be used that enrichrecipient cells within a cell population. For example, Type II callusdevelopment, followed by manual selection and culture of friable,embryogenic tissue, generally results in an enrichment of cells. Manualselection techniques which can be employed to select target cells mayinclude, e.g., assessing cell morphology and differentiation, or may usevarious physical or biological means. Cryopreservation also is apossible method of selecting for recipient cells.

A. Altering Gene Expression in a Plant

In accordance with the invention, alteration of expression of a gene asdescribed herein may comprise increasing expression of a gene, ordecreasing expression of a gene. As described herein, the presentinvention may comprise altering expression of a BADC gene. In someembodiments, methods are provided comprising completely silencing ordown-regulating expression of a gene. In other embodiments, partial orincomplete silencing or down-regulation of a gene may be sufficient toachieve the desired effect.

Alteration of gene expression in a plant may be accomplished by avariety of methods known in the art. In accordance with the invention,any method useful for altering expression of a gene or gene product maybe used, including, but not limited to, antisense, RNAi, CRISPR, TALON,nanobodies, EMS, T-DNA or transposon-mediated gene knockout, orconventional mutagenesis/targeted breeding. Such methods are known inthe art. As used herein the words “gene suppression” are intended torefer to any of the well-known methods for reducing the levels ofprotein produced as a result of gene transcription to mRNA andsubsequent translation of the mRNA.

Gene suppression is also intended to mean the reduction of proteinexpression from a gene or a coding sequence includingpost-transcriptional gene suppression and transcriptional suppression.Post-transcriptional gene suppression is mediated by the homologybetween of all or a part of a mRNA transcribed from a gene or codingsequence targeted for suppression and the corresponding double strandedRNA used for suppression, and refers to the substantial and measurablereduction of the amount of available mRNA available in the cell forbinding by ribosomes. The transcribed RNA may be in the senseorientation to effect what is called co-suppression, in the anti-senseorientation to effect what is called anti-sense suppression, or in bothorientations, in which case a dsRNA may be produced to achieve RNAinterference (RNAi). Such methods may be useful in accordance with theinvention for down-regulating or silencing a BADC gene as describedherein. Transcriptional suppression is mediated by the presence in thecell of a dsRNA, a gene suppression agent, exhibiting substantialsequence identity to a DNA sequence or the complement thereof to resultin promoter trans-suppression. Gene suppression may be effective againsta native gene associated with a trait, e.g., to produce a plant withreduced levels of a protein encoded by the native gene or with enhancedor reduced levels of an affected gene product. A gene product mayinclude an RNA molecule, including, but not limited to, mRNA, rRNA,tRNA, siRNA, shRNA, or the like. A gene product may also include aprotein or polypeptide, or a fragment thereof.

Post-transcriptional gene suppression by anti-sense or sense-orientedRNA to regulate gene expression in plant cells is known in the art, asis the use of dsRNA to suppress genes in plants. Post-transcriptionalgene suppression in plants may employ both sense-oriented andanti-sense-oriented, transcribed RNA that is stabilized, e.g., as ahairpin or stem-and-loop structure.

As used herein, the term “expression” refers to the transcription andstable accumulation of sense or anti-sense RNA derived from a nucleicacid. “Expression” may also refer to translation of mRNA into apolypeptide or protein. As used herein, the term “antisense RNA” refersto an RNA transcript that is complementary to all or a part of a mRNAthat is normally produced in a cell. The complementarity of an antisenseRNA may be with any part of the specific gene transcript, i.e., at the5′ non-coding sequence, 3′ non-translated sequence, introns, or thecoding sequence. As used herein, the term “RNA transcript” refers to theproduct resulting from RNA polymerase-catalyzed transcription of a DNAsequence. When the RNA transcript is a perfect complementary copy of theDNA sequence, it is referred to as the primary transcript or it may bean RNA sequence derived from post-transcriptional processing of theprimary transcript and is referred to as the mature RNA.

As used herein, the phrase “inhibition of gene expression” or“inhibiting expression of a target gene” refers to the absence (orobservable decrease) in the level of protein and/or mRNA product fromthe target gene. Specificity refers to the ability to inhibit the targetgene without manifest effects on other genes of the cell and without anyeffects on any gene within the cell that is producing the dsRNAmolecule. The inhibition of gene expression of a target gene asdescribed herein may result in novel phenotypic traits in the plant.

III. Production and Characterization of Stably Transformed Plants

After effecting delivery of exogenous DNA to recipient cells, the nextsteps generally concern identifying the transformed cells for furtherculturing and plant regeneration. In order to improve the ability toidentify transformants, one may desire to employ a selectable orscreenable marker gene with a transformation vector prepared inaccordance with the invention. In this case, one would then generallyassay the potentially transformed cell population by exposing the cellsto a selective agent or agents, or one would screen the cells for thedesired marker gene trait.

It is believed that DNA is introduced into only a small percentage oftarget cells in any one study. In order to provide an efficient systemfor identification of those cells receiving DNA and integrating it intotheir genomes one may employ a means for selecting those cells that arestably transformed. One exemplary embodiment of such a method is tointroduce, into the host cell, a marker gene which confers resistance tosome normally inhibitory agent, such as an antibiotic or herbicide.Examples of antibiotics which may be used include the aminoglycosideantibiotics neomycin, kanamycin and paromomycin, or the antibiotichygromycin. Resistance to the aminoglycoside antibiotics is conferred byaminoglycoside phosphostransferase enzymes such as neomycinphosphotransferase II (NPT II) or NPT I, whereas resistance tohygromycin is conferred by hygromycin phosphotransferase.

Potentially transformed cells then are exposed to the selective agent.In the population of surviving cells will be those cells where,generally, the resistance-conferring gene has been integrated andexpressed at sufficient levels to permit cell survival. Cells may betested further to confirm stable integration of the exogenous DNA.

One herbicide which constitutes a desirable selection agent is thebroad-spectrum herbicide bialaphos. Another example of a herbicide whichis useful for selection of transformed cell lines in the practice of theinvention is the broad-spectrum herbicide glyphosate. Glyphosateinhibits the action of the enzyme EPSPS which is active in the aromaticamino acid biosynthetic pathway. Inhibition of this enzyme leads tostarvation for the amino acids phenylalanine, tyrosine, and tryptophanand secondary metabolites derived therefrom. U.S. Pat. No. 4,535,060describes the isolation of EPSPS mutations which confer glyphosateresistance on the EPSPS of Salmonella typhimurium, encoded by the genearoA. The EPSPS gene from Zea mays was cloned and mutations similar tothose found in a glyphosate resistant aroA gene were introduced invitro. Mutant genes encoding glyphosate resistant EPSPS enzymes aredescribed in, for example, International Patent WO 97/4103.

To use the bar-bialaphos or the EPSPS-glyphosate selective system,transformed tissue is cultured for 0-28 days on nonselective medium andsubsequently transferred to medium containing from 1-3 mg/1 bialaphos or1-3 mM glyphosate as appropriate. While ranges of 1-3 mg/1 bialaphos or1-3 mM glyphosate will typically be preferred, it is proposed thatranges of 0.1-50 mg/1 bialaphos or 0.1-50 mM glyphosate will findutility.

Cells that survive the exposure to the selective agent, or cells thathave been scored positive in a screening assay, may be cultured in mediathat supports regeneration of plants. In an exemplary embodiment, MS andN6 media may be modified by including further substances such as growthregulators. One such growth regulator is dicamba or 2,4-D. However,other growth regulators may be employed, including NAA, NAA+2,4-D orpicloram. Media improvement in these and like ways has been found tofacilitate the growth of cells at specific developmental stages. Tissuemay be maintained on a basic media with growth regulators untilsufficient tissue is available to begin plant regeneration efforts, orfollowing repeated rounds of manual selection, until the morphology ofthe tissue is suitable for regeneration, at least 2 weeks, thentransferred to media conducive to maturation of embryoids. Cultures aretransferred every 2 weeks on this medium. Shoot development will signalthe time to transfer to medium lacking growth regulators.

The transformed cells, identified by selection or screening and culturedin an appropriate medium that supports regeneration, will then beallowed to mature into plants. Developing plantlets are transferred tosoilless plant growth mix, and hardened, e.g., in an environmentallycontrolled chamber, for example, at about 85% relative humidity, 600 ppmCO₂, and 25-250 microeinsteins m⁻² s⁻¹ of light. Plants may be maturedin a growth chamber or greenhouse. Plants can be regenerated in fromabout 6 weeks to 10 months after a transformant is identified, dependingon the initial tissue. During regeneration, cells are grown on solidmedia in tissue culture vessels. Illustrative embodiments of suchvessels are Petri dishes and Plant Cons. Regenerating plants can begrown at about 19 to 28° C. After the regenerating plants have reachedthe stage of shoot and root development, they may be transferred to agreenhouse for further growth and testing.

To confirm the presence of the exogenous DNA or “transgene(s)” in theregenerating plants, a variety of assays may be performed. Such assaysinclude, for example, “molecular biological” assays, such as Southernand northern blotting and PCR™; “biochemical” assays, such as detectingthe presence of a protein product, e.g., by immunological means (ELISAsand western blots) or by enzymatic function; plant part assays, such asleaf or root assays; and also, by analyzing the phenotype of the wholeregenerated plant.

Positive proof of DNA integration into the host genome and theindependent identities of transformants may be determined using thetechnique of Southern hybridization. Using this technique specific DNAsequences that were introduced into the host genome and flanking hostDNA sequences can be identified. Hence the Southern hybridizationpattern of a given transformant serves as an identifying characteristicof that transformant. In addition it is possible through Southernhybridization to demonstrate the presence of introduced genes in highmolecular weight DNA, i.e., confirm that the introduced gene has beenintegrated into the host cell genome. The technique of Southernhybridization provides information that is obtained using PCR™, e.g.,the presence of a gene, but also demonstrates integration into thegenome and characterizes each individual transformant.

Both PCR™ and Southern hybridization techniques can be used todemonstrate transmission of a transgene to progeny. In most instancesthe characteristic Southern hybridization pattern for a giventransformant will segregate in progeny as one or more Mendelian genes(Spencer et al., 1992) indicating stable inheritance of the transgene.

Whereas DNA analysis techniques may be conducted using DNA isolated fromany part of a plant, RNA will only be expressed in particular cells ortissue types and hence it will be necessary to prepare RNA for analysisfrom these tissues. PCR™ techniques also may be used for detection andquantitation of RNA produced from introduced genes. In this applicationof PCR™ it is first necessary to reverse transcribe RNA into DNA, usingenzymes such as reverse transcriptase, and then through the use ofconventional PCR™ techniques amplify the DNA. In most instances PCR™techniques, while useful, will not demonstrate integrity of the RNAproduct. Further information about the nature of the RNA product may beobtained by Northern blotting. This technique will demonstrate thepresence of an RNA species and give information about the integrity ofthat RNA. The presence or absence of an RNA species also can bedetermined using dot or slot blot northern hybridizations. Thesetechniques are modifications of northern blotting and will onlydemonstrate the presence or absence of an RNA species.

The expression of a gene product is often determined by evaluating thephenotypic results of its expression. These assays also may take manyforms including but not limited to analyzing changes in the chemicalcomposition, morphology, or physiological properties of the plant.Chemical composition may be altered by expression of genes encodingenzymes or storage proteins which change amino acid composition and maybe detected by amino acid analysis, or by enzymes that change starchquantity which may be analyzed by near infrared reflectancespectrometry. Morphological changes may include greater stature orthicker stalks. Most often changes in response of plants or plant partsto imposed treatments are evaluated under carefully controlledconditions termed bioassays.

IV. Breeding Plants of the Invention

In addition to direct transformation of a particular plant genotype witha construct prepared according to the current invention, transgenicplants may be made by crossing a plant having a selected DNA of theinvention to a second plant lacking the construct. For example, aselected BADC coding sequence can be introduced into a particular plantvariety by crossing, without the need for ever directly transforming aplant of that given variety. Therefore, the current invention not onlyencompasses a plant directly transformed or regenerated from cells whichhave been transformed in accordance with the current invention, but alsothe progeny of such plants. As used herein, the term “progeny” denotesthe offspring of any generation of a parent plant prepared in accordancewith the instant invention, wherein the progeny comprises a selected DNAconstruct prepared in accordance with the invention. “Crossing” a plantto provide a plant line having one or more added transgenes relative toa starting plant line, as disclosed herein, is defined as the techniquesthat result in a transgene of the invention being introduced into aplant line by crossing a plant of a starting line with a plant of adonor plant line that comprises a transgene of the invention. To achievethis one could, for example, perform the following steps:

(a) plant seeds of the first (starting line) and second (donor plantline that comprises a transgene of the invention) parent plants;

(b) grow the seeds of the first and second parent plants into plantsthat bear flowers;

(c) pollinate a flower from the first parent plant with pollen from thesecond parent plant; and

(d) harvest seeds produced on the parent plant bearing the fertilizedflower.

Backcrossing is herein defined as the process including the steps of:

(a) crossing a plant of a first genotype containing a desired gene, DNAsequence or element to a plant of a second genotype lacking the desiredgene, DNA sequence or element;

(b) selecting one or more progeny plant containing the desired gene, DNAsequence or element;

(c) crossing the progeny plant to a plant of the second genotype; and

(d) repeating steps (b) and (c) for the purpose of transferring adesired DNA sequence from a plant of a first genotype to a plant of asecond genotype.

Introgression of a DNA element into a plant genotype is defined as theresult of the process of backcross conversion. A plant genotype intowhich a DNA sequence has been introgressed may be referred to as abackcross converted genotype, line, inbred, or hybrid. Similarly a plantgenotype lacking the desired DNA sequence may be referred to as anunconverted genotype, line, inbred, or hybrid.

V. Definitions

As used herein, accessions AT3G56130, AT1G52670, and AT3G15690 areintended to refer to BADC1, BADC2, and BADC3, respectively.

As used herein, α-CT refers to AT2G38040; β-CT refers to ATCG00500; BCrefers to AT5G35360; BCCP1 refers to AT5G16390; BCCP2 refers toAT5G15530; BADC1 refers to AT3G56130; BADC2 refers to AT1G52670; andBADC3 refers to AT3G15690.

Endogenous: A sequence natively found in a host cell or a cell of thesame species. In one embodiment, an endogenous sequence may beoverexpressed or expressed at a higher level compared to wildtype andstill be considered endogenous.

Expression: The combination of intracellular processes, includingtranscription and translation, undergone by a coding DNA molecule suchas a structural gene to produce a polypeptide.

Genetic Transformation: A process of introducing a DNA sequence orconstruct (e.g., a vector or expression cassette) into a cell orprotoplast in which that exogenous DNA is incorporated into a chromosomeor is capable of autonomous replication.

Heterologous: A sequence which is not normally present in a given hostgenome in the genetic context in which the sequence is currently found.In this respect, the sequence may be native to the host genome, but berearranged with respect to other genetic sequences within the hostsequence. For example, a regulatory sequence may be heterologous in thatit is linked to a different coding sequence relative to the nativeregulatory sequence. In addition, a particular sequence can be“heterologous” with respect to a cell or organism into which it isintroduced (for example, a sequence that does not naturally occur inthat particular cell or organism).

Obtaining: When used in conjunction with a transgenic plant cell ortransgenic plant, obtaining means either transforming a non-transgenicplant cell or plant to create the transgenic plant cell or plant, orplanting transgenic plant seed to produce the transgenic plant cell orplant. Such a transgenic plant seed may be from an R₀ transgenic plantor may be from a progeny of any generation thereof that inherits a giventransgenic sequence from a starting transgenic parent plant.

Promoter: A recognition site on a DNA sequence or group of DNA sequencesthat provides an expression control element for a structural gene and towhich RNA polymerase specifically binds and initiates RNA synthesis(transcription) of that gene.

R₀ transgenic plant: A plant that has been genetically transformed orhas been regenerated from a plant cell or cells that have beengenetically transformed.

Regeneration: The process of growing a plant from a plant cell (e.g.,plant protoplast, callus, or explant).

Selected DNA: A DNA segment which one desires to introduce or hasintroduced into a plant genome by genetic transformation.

Transformation construct: A chimeric DNA molecule designed forintroduction into a host genome by genetic transformation. Preferredtransformation constructs will comprise all of the genetic elementsnecessary to direct the expression of one or more exogenous genes. Inparticular embodiments of the instant invention, it may be desirable tointroduce a transformation construct into a host cell in the form of anexpression cassette.

Transformed cell: A cell in which the DNA complement has been altered bythe introduction of an exogenous DNA molecule into that cell.

Transgene: A segment of DNA which has been incorporated into a hostgenome or is capable of autonomous replication in a host cell and iscapable of causing the expression of one or more coding sequences.Exemplary transgenes will provide the host cell, or plants regeneratedtherefrom, with a novel phenotype relative to the correspondingnon-transformed cell or plant. Transgenes may be directly introducedinto a plant by genetic transformation, or may be inherited from a plantof any previous generation which was transformed with the DNA segment.

Transgenic plant: A plant or progeny plant of any subsequent generationderived therefrom, wherein the DNA of the plant or progeny thereofcontains an introduced exogenous DNA segment not naturally present in anon-transgenic plant of the same strain. The transgenic plant mayadditionally contain sequences which are native to the plant beingtransformed, but wherein the “exogenous” gene has been altered in orderto alter the level or pattern of expression of the gene, for example, byuse of one or more heterologous regulatory or other elements.

Vector: A DNA molecule designed for transformation into a host cell.Some vectors may be capable of replication in a host cell. A plasmid isan exemplary vector, as are expression cassettes isolated therefrom.

The description herein is merely exemplary in nature and, thus,variations that do not depart from the gist of that which is describedare intended to be within the scope of the teachings. Such variationsare not to be regarded as a departure from the spirit and scope of theteachings.

EXAMPLES

The following examples are included to demonstrate preferred embodimentsof the invention. It should be appreciated by those of skill in the artthat the techniques disclosed in the examples, which follow representtechniques discovered by the inventor to function well in the practiceof the invention, and thus can be considered to constitute preferredmodes for its practice. However, those of skill in the art should, inlight of the present disclosure, appreciate that many changes can bemade in the specific embodiments, which are disclosed and still obtain alike or similar result without departing from the spirit and scope ofthe invention.

Example 1

Two Novel Proteins, BADC1 and BADC2, Co-Immunoprecipitate with Hetaccase

To discover unknown protein interactors with the hetACCase, quantitativeco-innmunoprecipitation (co-IP) analyses were performed. Wild type A.thaliana (ecotype-Columbia-0) were grown in a growth chamber withlong-day (16 h, 23° C., 50% humidity, 50 pmol m⁻² s⁻¹) and short-night(8 h, 20° C., 50% humidity) conditions. For co-immunoprecipitationstudies, 8.5 cm×8.5 cm pots were filled with moist soil (Sunshine Mix#6, Sun Gro Horticulture), covered with screen (1 mm² pore size), andcoated with seeds.

For co-innmunoprecipitation of hetACCase from Arabidopsis seedlings,crude chloroplasts were isolated from approximately 10 g 14-d-old A.thaliana seedlings after 1 h light exposure. Fresh leaves werehomogenized in ice-cold grinding buffer (50 mM HEPES-KOH pH 8.0, 330 mMsorbitol, 1.5 mM MnCl₂, 2 mM MgCl₂, 2 mM EDTA, 0.1% (w/v) BSA) using aWaring blender. Homogenate was filtered through two layers of Miraclothand centrifuged at 2,600 g at 4° C. for 20 min. Chloroplasts were lysedfor 30 min in ice-cold lysis buffer (50 mM HEPES-KOH pH 8.0, 10% (v/v)glycerol, 0.5% (v/v) Triton X-100). Lysates were homogenized ten timesin a Dounce homogenizer on ice and then centrifuged at 30 k g for 20 minat 4° C. Then, 1 mL of the 30 k g supernatant was added to 25 μL ProteinA-Sepharose beads (Sigma Aldrich) either uncoated (control) or coatedwith antibody specific for α-CT, BCCP, BADC1, or BADC2.Co-immunoprecipations (co-IP) were carried out at 4° C. for 3 h withgentle mixing. The beads were washed twice with 1 mL ice-cold lysisbuffer and precipitated protein was eluted by adding 30 μL 6× SDS samplebuffer (350 mM Tris-HCl, pH 6.8, 350 mM SDS, 30% (v/v) glycerol, 100 mMdithiothreitol, 2.5 mM bromophenol blue) and heating at 65° C. for 10min. Eluted proteins were resolved on 10% SDS-PAGE gels for western andmass spectrometry analysis.

As described in detail above, clarified chloroplast lysates from14-d-old A. thaliana seedlings were incubated with Protein A-Sepharosebeads coated with polyclonal antibodies to either BCCP2 or α-CT. Controlprecipitations were performed using uncoated beads. Precipitatedproteins were identified by LC-MS/MS analysis of trypsin-digestedpeptides.

For mass spectrometry, sample preparation and analysis was as follows.Precipitated proteins from co-IPs were resolved by 10% SDS-PAGE andstained with colloidal Coomassie Brilliant Blue (CBB) G-250. Each lanewas separated into 0.5 cm segments and subsequently diced intoapproximately 1 mm³ gel pieces. Gel pieces were digested withsequencing-grade trypsin and peptides were extracted according tomethods known in the art. Tryptic peptides were lyophilized and storedat −20° C. until analysis by liquid chromatography-tandem massspectrometry (LC-MS/MS). Lyophilized peptides were prepared for massspectrometry analysis as described previously. Samples were analyzed ona LTQ Orbitrap XL ETD (Thermo Fisher Scientific) according to Swatek etal., (Biochem J 459(1): 15-25, 2014), with the exception that peptideswere eluted using a 30 min acetonitrile gradient (5-43% acetonitrile),the top 8 masses from the precursor scan were selected fordata-dependent acquisition, and precursor ions were fragmented using CID(collision-induced dissociation). Dynamic exclusion was enabled with thefollowing settings: repeat count, 3; repeat duration, 30 s; exclusionlist, 50; and exclusion list duration, 30 s.

Acquired spectra were searched against the TAIR10 protein database(70,773 entries, downloaded on Jun. 11, 2012), concatenated to arandomized TAIR10 database as a decoy. Search parameter settings ofSEQUEST were static modification of cysteine-carboxyamidomethylation andvariable modification of methionine-oxidation. Other search parametersettings of SEQUEST included two missed tryptic cleavage sites, absolutethreshold: 1000, minimum ion count: 10, mass range: 650-3500, and aparent and fragment ion tolerance of 1 Da and 1000 ppm, respectively.Search result files were loaded into Proteome Discoverer 1.3 (ThermoFisher Scientific). Identified peptides were filtered to <1% falsediscovery rate using the following criteria: 10 ppm peptide massdeviation, ‘Xcorr versus charge state’, and 2 and 1 peptide minimum forco-IPs and 2D BN-SDS PAGE, respectively. Protein grouping was alsoenabled. False discovery rate was calculated manually using spectralcounting. Files generated for each biological replicate by ProteomeDiscoverer 1.3 were exported into Microsoft Excel for further analysis.

Proteins identified from SEQUEST searches were compared against uncoatedSepharose bead controls that had been treated in an identical manner tothe hetACCase subunit co-IPs. Proteins that were only identified in thehetACCase subunit co-IPs were considered as putative interactingclients. All other proteins were disregarded. The hetACCase subunitswere never identified in controls.

Quantitative mass spectrometry of these co-IPs revealed all four knownsubunits to ACCase and two unknown proteins annotated as ‘biotin/lipoylattachment domain containing’ (BADC) proteins. From seven biologicalreplicates of the α-CT co-IPs, the entire hetACCase complex wasidentified. Likewise, all subunits, except β-CT, were identified fromco-IPs with antibodies to BCCP2 (FIGS. 1A and 1B). As expected, theBC/BCCP and α/β-CT subcomplexes were relatively higher in abundance inthe BCCP2 and α-CT co-IPs, respectively. Additionally, two unknownproteins with a “biotin attachment domain-containing” region, hereaftertermed BADC1 (AT3G56130) and BADC2 (AT1G52670), were identified fromboth co-IPs. The BADC1 protein was present in seven and one replicate ofthe BCCP2 and α-CT co-IPs, respectively, while BADC2 was present in sixand two replicates of the BCCP2 and α-CT co-IPs, respectively. Thenormalized, relative abundance of these proteins was more commensuratewith BC and BCCP abundance than α- and β-CT from both co-IP analyses.Reciprocal co-IPs using antibodies specific to BADC1 and BADC2precipitated both BCCP isoforms (FIGS. 1C and 1D). Thus, BADC1 and BADC2appear to interact with the BC/BCCP components of hetACCase.

Example 2

Recombinant Protein Expression and Purification and Immunoblotting

The ORFs of BCCP1, BCCP2, BADC1, BADC2, and BADC3 were amplified via PCRfrom a cDNA clone (ABRC). The primer pairs for these amplifications werethe same as those used in the yeast two-hybrid construct formation (FIG.2). These primers were designed to remove the transit peptide, aspredicted by TargetP. The amplified ORF of all five genes were clonedinto either the expression vector pET28a or pET11a producing anN-terminal His-tagged fusion protein or an untagged recombinant protein,respectively. All constructs were sequence confirmed via DNA sequencing.Constructs were then transformed into E. coli strain BL21 (B2685:Sigma). Recombinant protein was expressed and purified from transformedBL21 cells as described in Swatek et al., J Proteome Res10(9):4076-4087, 2011). For co-expression studies, ˜200 ng of eachplasmid was used to transform BL21 cells.

Proteins resolved by SDS-PAGE were transferred to PVDF membrane andstained with the appropriate primary antibody overnight at 4° C. forwestern blot analysis. All antibodies were used at 1:5000 dilution inPBS-T (10 mM NaH₂PO₄—NaOH pH 7.2, 150 mM NaCl, 0.3% (v/v) Tween 20).hetACCase antibodies used in this study were derived from rabbitsimmunized with recombinant P. sativum α-CT, recombinant A. thalianaBCCP2, or recombinant A. thaliana BADC1. Blots were rinsed twice inPBS-T and probed in secondary antibody for 1 h at room temperature anddeveloped. Goat anti-rabbit IgG secondary antibody conjugated toalkaline phosphatase was obtained from Sigma-Aldrich (St. Louis, Mo.).

Example 3

Orthogonal Approaches Confirm a Direct Interaction Between Three BADCand Two BCCP Isoforms from Arabidopsis.

Yeast two-hybrid construct design. The ORF of genes of interest wereinserted into bait and prey vectors PGBKT7 and pGADT7. Primers weredesigned to exclude the transit peptide from the coding region, aspredicted by TargetP (FIG. 2). Genes were amplified from cDNA clonesobtained from the Arabidopsis Biological Resource Center Amplicons werefirst inserted into Zero Blunt TOPO vector (Life Technologies) andchecked for errors by DNA sequencing. Error-free amplicons were thensubcloned into either pGBKT7 or pGADT7 vector. Completed constructs weretransformed into competent DH5α cells. Cells transformed with pGBKT7 andpGADT7 were grown on LB media agar plates containing 50 pg/mL kanamycin(Kan) and 100 pg/mL ampicillin (Amp), respectively. Plasmids werepurified from 5 mL culture of positive colonies using Q1Aprep SpinMiniprep kit (Qiagen).

To confirm the co-IP results and determine which hetACCase subunitdirectly interacts with BADC1 and BADC2, targeted yeast two-hybridanalysis was employed using an adaptation of the lithium acetate method.Strain AH109 yeast were transformed with 100 ng of bait and prey vector.Pelleted transformed cells were resuspended in 300 μL sterile water.Aliquots of 100 μL cell suspension were plated on synthetic dropout (SD)media lacking leucine, tryptophan, and histidine. Plates were incubatedat 30° C. for 4 d and then imaged. Images shown are representative of atleast three replicate studies.

In addition to the two experimentally-identified BADCs a third, putativeBADC isoform was also tested, termed BADC3 (AT3G15690), identified byBLAST interrogation of the A. thaliana genome. This protein shares 61%amino acid identity with BADC2, suggesting it might have similarfunction. Based upon yeast two hybrid analyses, each of the three BADCproteins interacted with each BCCP isoform. As shown in FIG. 3A, strainAH109 yeast was transformed with bait and prey constructs containing thegenes shown. Negative controls showed minimal or no growth. Sv40 and p53were used as positive controls. Transformed yeast were plated on medialacking Trp, Leu, and His. Results shown are representative of threebiological replicates. Additionally, each of the BADC isoforms interactswith one another.

To further evaluate the interaction between BADC and BCCP, A. thalianaBCCP1 was co-expressed with each of the three A. thaliana BADC proteinsin E. coli. In these studies, either the BADC or BCCP1 protein wasexpressed with a His₆-tag, while the other contained no affinity tag.When the Hiss-tagged protein was purified by Ni²⁺-NTA affinitychromatography, the respective “untagged” protein was present in thesame elution fractions. In FIG. 3B, the Coomassie-stained gels show theelution fractions of Ni²⁺-NTA-purified protein from E. coli. At right, anative protein was co-expressed with a His₆-tagged protein. At left, thenative protein was expressed alone. The native proteins were presentstrongly in the elution fractions only when co-expressed with theHis₆-tagged protein. Protein identities were confirmed by LC-MS/MS.These studies were performed to test if the BADC proteins can interactdirectly with ACCase. The results confirm the BADC proteins associatewith ACCase via a direct interaction with the BCCP and BC subunits. As acontrol, it was verified that untagged proteins were unable to bind tothe affinity column. Using this system, it was observed that A. thalianaBC and BADC3 also co-purified, suggesting that BADC3, and likely BADC1and 2, may also interact with BC. These studies confirm BADC isoformsdirectly interact with the BCCP and BC subunits of hetACCase.

Example 4

Biotin is not Required for BADC-BCCP Interaction

To determine if the BCCP-BADC interaction involves the biotin cofactor,as previously reported for PII interaction with hetACCase, the biotinylLys245 residue on BCCP1 was mutated to Arg by site-directed mutagenesis.This mutation prevents biotinylation of BCCP1. Using this ‘apo-BCCP1’,the yeast two-hybrid and co-expression analysis was repeated with BADCs.All BADC isoforms were shown to interact with apo-BCCP1.

Example 5

Recombinant AtBADC1 and AtBADC3 Form Homodimers through a Disulfide Bond

Previous analysis of E. coli BCCP suggested that this subunit formsfunctional homodimers in vivo. Through intact mass analysis of purifiedrecombinant BCCP2, it was observed that plant BCCP can also formhomodimers. In addition, analysis of recombinant BADCs showed that BADC1and BADC3, but not BADC2, can form homodimers. The observed monomer sizefor each BADC was in agreement with the predicted mass, suggesting theseproteins are unmodified. In the absence of DTT, purified recombinantBCCP2, BADC1, and BADC3 show a monomer and dimer band when denatured andresolved by SDS-PAGE. Recombinant BADC2 shows only a monomer band.Increasing DTT concentration led to the disappearance of the dimer band,suggesting a disulfide bond is involved in dimer formation of BADCs andplant BCCPs.

Example 6

BADCs Resemble BCCPs but are not Biotinylated.

The three BADC isoforms share many characteristics with the two BCCPisoforms from A. thaliana. First, these proteins contain a canonicalplastid target peptide and are plastid localized based uponbioinformatic predictions, co-IP, and western blot studies. Secondly,the BADC isoforms share 24 to 29% amino acid identity with the BCCPisoforms (FIG. 4). Last, structural predictions of the BADC and BCCPproteins (FIG. 5) show similar (3-sheet secondary structure with acharacteristic “thumb motif” as previously observed for the E. coliBCCP. Intact mass analysis showed that BADC proteins are able to formhomodimers.

Despite these similarities, the BADC proteins lack the canonicalbiotinylation motif. As shown in FIG. 6A, the alignment of the C-terminiof the Arabidopsis thaliana BCCP1 and BCCP2 and BADC proteins (BADC1,BADC2, and BADC3) shows multiple conserved residues (shown in bold). Thecanonical biotinylation motif containing the biotinyl Lys in BCCP1/2 isshown boxed in on lines 1 and 2 of the sequence alignment. The alignmentcomparison indicates the BADC proteins resemble BCCP isoforms but lack acanonical biotinylation motif. The tetrapeptide(AlaNal)-Met-Lys-(Met/Leu) is the known biotinylation motif and isusually located 34 or 35 residues from the C-terminus. The BADC proteinsdo, however, possess a conserved Lys residue in a similar(Val/Ile)-(LeuNal)-Lys-(Leu/Ile) motif located near the C-terminussuggesting the possibility of a non-canonical biotinylation motif.

To test this possibility, recombinant BADC proteins expressed in E. coliand the native BADC1 protein from A. thaliana seedlings were purifiedand probed for biotinylation using a biotin-specific antibody. As shownin FIG. 6B, based on the western blotting analysis of recombinantArabidopsis proteins using a biotin-specific antibody, BCCP2 is observedto be biotinylated, while the BADCs are not. As shown in FIG. 6C, whichis the protein blot analysis of immunoprecipitated in vivo BADC1 fromArabidopsis seedlings, blotting precipitate with BADC1-specific antibodyshows the presence BADC1 in the sample, while blotting withbiotin-specific antibody shows no recognition of BADC1. Results of thesestudies confirmed that the BADC proteins are not biotinylated in vivo,although BCCP controls clearly were.

Example 7

BADC Orthologs are Present in Green Algae and Land Plants but notBacteria.

Identification of BADC orthologs and co-occurrence analysis suggestsBADCs first appeared in red algae. The evidence of a direct BADC-BCCPinteraction suggests that BADC function is linked to hetACCase. If true,orthologs to A. thaliana BADCs (AtBADC) would be expected to reside onlyin organisms that contain hetACCase, not the homomeric form thatpredominates in eukaryotes. To search for the presence of AtBADCorthologous proteins, the primary sequence of each AtBADC was used tosearch against the KEGG Sequence Similarity database. Putative orthologswere confirmed by reciprocal BLAST searches against the A. thalianaproteome. All AtBADC orthologs lacked the conserved biotinyl Lys foundin BCCPs. Orthologous proteins were identified for AtBADCs across 78different species of land plants and algae (Table 1). The full-lengthprotein sequences of identified AtBADC orthologs were used to generate amaximum-likelihood phylogenetic tree. All of the species that harbor aputative AtBADC ortholog also contain the heteromeric form of ACCase. Noorthologs were detected in organisms that contain only the homomericACCase. Additionally, no AtBADC orthologs were detected in prokaryotes,which also contain a hetACCase. The presence of orthologs in algae butnot prokaryotes suggests that BADCs first appeared in algae.

TABLE 1 Orthologous proteins identified for AtBADC1, AtBADC2, andAtBADC3. Species Category BCCP1 BCCP2 BADC1 BADC2 BADC3 Amborellatrichopoda Flowering XP_011621081.1 N/A XP_011627066.1 N/AXP_011622803.1 Plants Arabidopsis lyrata subsp. lyrata EudicotsXP_002873773.1 XP_002871669.1 XP_002876350.1 XP_002894393.1XP_002882958.1 Arabidopsis thaliana Eudicots NP_197143.1 NP_568316.1NP_567035.1 NP_564612.1 NP_188190.1 Arabis alpina Eudicots KFK25879.1KFK25777.1 KFK34856.1 N/A KFK38917.1 Arachis duranensis EudicotsXP_015962701.1 XP_015946097.1 XP_015944188.1 N/A XP_015933506.1 Arachisipaensis Eudicots XP_016194346.1 XP_016181644.1 XP_016181047.1 N/AXP_016170604.1 Auxenochlorella protothecoides Green Algae XP_011398894.1N/A N/A N/A XP_011395766.1 Beta vulgaris subsp. vulgaris EudicotsXP_010679318.1 N/A XP_010692910.1 N/A XP_010691182.1 Brachypodiumdistachyon Monocots N/A N/A N/A N/A N/A Brassica napus EudicotsXP_013663621.1 XP_013728269.1 XP_013663447.1 N/A XP_013645085.1 Brassicaoleracea var. oleracea Eudicots XP_013625183.1 XP_013621850.1XP_013605292.1 N/A XP_013585896.1 Brassica rapa Eudicots XP_009131537.1XP_009131471.1 XP_009116310.1 N/A XP_009115305.1 Cajanus cajan EudicotsKYP60383.1 N/A KYP44948.1 N/A KYP59593.1 Camelina sativa EudicotsXP_010453799.1 XP_010453692.1 XP_010504497.1 XP_010479781.1XP_010465548.1 Capsella rubella Eudicots XP_006286538.1 XP_006288243.1N/A XP_006305526.1 XP_006298142.1 Capsicum annuum EudicotsXP_016573862.1 N/A XP_016575440.1 N/A XP_016578500.1 Chlamydomonasreinhardtii Green Algae N/A XP_001700442.1 N/A N/A XP_001690119.1Chlorella variabilis Green Algae N/A XP_005850451.1 N/A N/AXP_005845403.1 Cicer arietinum Eudicots XP_012569122.1 N/AXP_004500525.1 N/A XP_004486692.1 Citrus clementina Eudicots N/AXP_006431277.1 XP_006435833.1 N/A XP_006427204.1 Citrus sinensisEudicots N/A XP_006482733.1 XP_006486239.1 N/A XP_006465373.1 Coccomyxasubellipsoidea C-169 Green Algae N/A XP_005649768.1 N/A N/AXP_005646014.1 Cucumis melo Eudicots N/A XP_008456473.1 XP_008441486.1N/A XP_008461084.2 Cucumis sativus Eudicots N/A XP_004137199.1XP_011656420.1 N/A XP_004135840.1 Daucus carota subsp. sativus EudicotsKZM82431.1 N/A KZM80059.1 N/A KZM88409.1 Dorcoceras hygrometricumEudicots KZV23283.1 N/A KZV16809.1 N/A N/A Elaeis guineensis MonocotsXP_010936329.1 N/A XP_010938420.1 N/A XP_010921048.1 Erythranthe guttataEudicots XP_012834625.1 XP_012844390.1 XP_012858601.1 N/A XP_012856758.1Eucalyptus grandis Eudicots XP_010038361.1 XP_010032526.1 XP_010067567.1N/A XP_010033940.1 Eutrema salsugineum Eudicots XP_006400177.1XP_006400073.1 XP_006403020.1 XP_006392894.1 XP_006406933.1 Fragariavesca subsp. vesca Eudicots N/A XP_004304236.1 XP_004307696.1 N/AXP_004302964.1 Genlisea aurea Eudicots EPS63946.1 N/A N/A EPS63437.1 N/AGlycine max Eudicots N/A XP_003543944.1 XP_006590336.1 N/AXP_003543673.1 Glycine soja Eudicots KHN13569.1 N/A KHN04794.1 N/AKHN44161.1 Gonium pectorale Green Algae N/A KXZ51015.1 N/A N/AKXZ55249.1 Gossypium arboreum Eudicots KHG03380.1 KHG02691.1 N/AKHG02291.1 N/A Gossypium hirsutum Eudicots XP_016683408.1 XP_016752201.1XP_016724217.1 N/A N/A Gossypium raimondii Eudicots XP_012451021.1 N/AXP_012462883.1 N/A XP_012454990.1 Helicosporidium sp. ATCC 50920 GreenAlgae KDD76354.1 N/A N/A N/A KDD73528.1 Jatropha curcas EudicotsXP_012085783.1 XP_012084810.1 XP_012086589.1 N/A XP_012073227.1Klebsormidium flaccidum Green Plants N/A GAQ84037.1 N/A N/A GAQ80014.1Malus domestica Eudicots N/A XP_008379410.1 XP_008374383.1 N/A N/AMarchantia polymorpha subsp. Liverworts OAE20385.1 N/A N/A N/AOAE28621.1 polymorpha Medicago truncatula Eudicots XP_003624197.1 N/AXP_003616717.1 N/A XP_003597852.2 Morus notabilis Eudicots N/AXP_010089617.1 XP_010087032.1 XP_010097264.1 N/A Musa acuminata subsp.malaccensis Monocots XP_009394324.1 N/A XP_009418932.1 N/A N/A Nelumbonucifera Eudicots XP_010259375.1 N/A XP_010250846.1 N/A XP_010254348.1Nicotiana sylvestris Eudicots XP_009759359.1 N/A XP_009785832.1XP_009787427.1 N/A Nicotiana tabacum Eudicots XP_016465895.1XP_016481002.1 XP_016473105.1 N/A XP_016514334.1 Nicotianatomentosiformis Eudicots XP_009588294.1 XP_009616596.1 XP_009618778.1N/A XP_009628995.1 Phaseolus vulgaris Eudicots XP_007139713.1 N/AXP_007163588.1 N/A XP_007150717.1 Phoenix dactylifera MonocotsXP_008805110.1 N/A XP_008789922.1 N/A XP_008809725.1 Physcomitrellapatens Mosses N/A XP_001754932.1 N/A N/A XP_001775667.1 Populuseuphratica Eudicots XP_011013398.1 XP_011013434.1 XP_011004753.1 N/AXP_011040023.1 Populus trichocarpa Eudicots N/A XP_002305399.1XP_002311250.1 N/A XP_002299605.2 Prunus mume Eudicots XP_008240458.1N/A XP_008233825.1 XP_008228653.1 N/A Prunus persica EudicotsXP_007204703.1 XP_007215770.1 XP_007218764.1 XP_007215787.1 N/A Pyrus ×bretschneideri Eudicots N/A XP_009360536.1 XP_009369234.1 XP_009349124.1XP_009348645.1 Ricinus communis Eudicots XP_015572257.1 XP_002526099.1XP_002520803.1 N/A XP_015573743.1 Selaginella moellendorffii Club-MossesXP_002963883.1 N/A N/A N/A XP_002963889.1 Sesamum indicum Eudicots N/AXP_011072842.1 XP_011084859.1 N/A XP_011072247.1 Setaria italicaMonocots N/A N/A N/A N/A N/A Solanum lycopersicum EudicotsNP_001234322.1 N/A XP_004240889.1 N/A XP_004241703.1 Solanum pennelliiEudicots XP_015076155.1 N/A XP_015080112.1 N/A XP_015079819.1 Solanumtuberosum Eudicots XP_006345777.1 N/A XP_006353414.1 N/A XP_006356200.1Sorghum bicolor Monocots N/A N/A N/A N/A N/A Spinacia oleracea EudicotsKNA11791.1 N/A KNA11168.1 N/A KNA24821.1 Tarenaya hassleriana EudicotsXP_010558581.1 XP_010551815.1 XP_010534633.1 XP_010535127.1 N/ATheobroma cacao Eudicots N/A XP_007029252.1 XP_007008844.1 N/AXP_007023903.1 Triticum urartu Monocots N/A N/A N/A N/A N/A Vignaangularis Eudicots N/A KOM56589.1 KOM39631.1 N/A KOM44575.1 Vignaradiata var. radiata Eudicots N/A XP_014523207.1 XP_014494474.1 N/AXP_014498647.1 Vitis vinifera Eudicots XP_010649227.1 XP_002284374.1XP_002278151.2 N/A XP_002285378.1 Volvox carteri f. nagariensis GreenAlgae N/A XP_002952670.1 N/A N/A XP_002954026.1 Zea mays Monocots N/AN/A N/A N/A N/A Ziziphus jujuba Eudicots XP_015875754.1 XP_015879793.1XP_015877502.1 N/A XP_015868335.1 Zostera marina Monocots N/A KMZ60645.1KMZ56653.1 KMZ55983.1 N/A Galdieria sulphuraria Red Algae N/AYP_009051081.1 XP_005708748.1 N/A N/A

To determine if BADCs arose from a previously functional BCCP in algae,co-occurrence analysis was performed. With the exception of two redalgae and Cyanophora paradoxa all species contained AtBCCP and AtBADCorthologs. In red algae, only one putative AtBADC1 ortholog (GenBank ID:XP_005708748.1) was identified in the species Galdieria sulphuraria.This protein shares the same number of identical (31) and similar (46)amino acid residues with both AtBADC1 and AtBADC2, as well as 30identical and 44 similar amino acid residues with AtBADC3. However, theBLAST search attributed the highest score to AtBADC1. In addition, twoputative BCCP proteins were identified in the red algae species Chondruscrispus and Cyanidioschyzon merolae to lack the biotin motif residue butshared higher sequence similarity to AtBCCP2 than AtBADCs (GenBank IDXP_005715802.1 and XP_005535248.1, respectively), suggesting that BADCsoriginated from a BCCP gene duplication and loss-of-function mutation inred algae. From this observation, it appears not only that BADCs andBCCPs are related, but that the branch point between these proteinsoccurred in red algae, particularly since the more primitiveglaucophytes contain no AtBADC orthologs.

If BADC proteins are exclusive to acetyl-CoA carboxylases and not otheracyl-CoA carboxylases, BADC protein orthologs would be found only inplant clades that contain hetACCase. To search for BADCortholog-containing species, PSI-BLAST was performed using the primarysequence of each BADC from A. thaliana. Putative orthologs wereconfirmed by reciprocal BLAST searches against the A. thaliana proteomeand all putative BADCs were manually confirmed to lack a conservedbiotinyl Lys. FIG. 7 shows the phylogenetic tree of species containingan ortholog to all three BADCs. Orthologs to the A. thaliana BADCs wereonly observed in dicots, non-graminaceous monocots, and green algae. Thefounding member of the family, BADC3, dates back to green algae. All ofthese plant clades contain a hetACCase. No BADC ortholog was detected inprokaryotes or graminaceous monocots, the latter containing only thehomomeric form of ACCase.

Example 8

BADC3 Expression Reduces hetACCase Activity in a Temperature-SensitiveE. coli Mutant

Due to their similarity with BCCPs but lack of a conserved biotinylationmotif, BADCs may be negative regulators of hetACCase activity. As E.coli contain hetACCase but lack BADC orthologs, this system wasappropriate to test this theory. In vivo growth assays in E. coli accbstrain L8 were performed to evaluate the potential the effect of theBADC proteins on hetACCase activity. This strain containstemperature-sensitive (Ts) mutations in the BCCP gene (accB) thatprevent de novo FAS. Thus, cell growth at 37° C. is directly correlatedto hetACCase activity when lacking an exogenous source of fatty acids.Experiments were performed in minimal media containing only glucose andglycerol as carbon sources. In brief, the temperature-sensitive (Ts) L8strain E. coli was obtained from the Coli Genetic Stock Center (Yale,New Haven, Conn.) and transformed with the vectors in the text using theheat shock method. Transformants were selected by antibiotic resistanceand confirmed by PCR. Prior to the growth experiment, cultures weregrown overnight in LB media at 30° C. Overnight cultures werecentrifuged at 3,000 g and resuspended in 5 mL sterile deionized water.Cultures were centrifuged again and resuspended in M63 minimal media tomake OD600=3.75. Then 200 μL cell suspension was added to 7 mL M63 mediaplus antibiotics in 15 mL sterile culture tubes. Cultures contained Kan,and Amp if necessary, at 50 μg/mL each as well as 1 μM isopropylβ-D-1-thiogalactopyranoside at T=0.

To complement the Ts phenotype, the native E. coli BCCP (EcBCCP) genewas cloned into L8 cells in the inducible pET28a vector. Inducedexpression of EcBCCP rescued cell growth at 37° C. in media lackingfatty acids, while empty vector controls showed minimal growth. In FIG.8A, the growth curves show the optical density of L8 E. coli cells overtime. Cultures of transformed L8 cells were grown in M63 liquid cultureat 37° C. in the absence of fatty acids. Transformed cells contained thefollowing vectors: empty pET28a (EV28a), pET28a containing the E. coliBCCP gene (EcBCCP), empty pET11a (EV11a), and/or pET11a containing theAtBADC3 gene (BADC3). At T=0 h, the optical density was 0.15 and proteinexpression was induced with 1 μM IPTG. The EV control cells show minimalgrowth at 37° C., while re-introduction of native EcBCCP complementedthe temperature-sensitive phenotype. BADC3 expression alone showed nostatistical difference from EV control except at T=10 h, whileco-expression of BADC3 with EcBCCP showed an approximate 75% reductionin growth compared to EcBCCP alone across multiple studies. Resultsshown are representative of three separate studies. Error bars representstandard deviation.

In the same way, the A. thaliana BADC3 gene was cloned into L8 cells andwas unable to complement the Ts phenotype. Co-expression of BADC3 withEcBCCP reduced the complementing effect of EcBCCP expression by 71 to74% over multiple replicates. Affinity pull-down assays with taggedBADC3 confirmed the inhibition was mediated by interaction with EcBCCP.FIG. 8B shows the Coomassie blue stain and anti-biotin protein blot ofNi²⁺-NTA purified His₆-BADC3. Protein blot shows that EcBCCP proteinco-purifies with His₆-BADC3, indicating its direct effect on ACCaseactivity. The experiment demonstrated that BADC protein expression has anegative effect on E. coli growth. Co-association of BADC3 with EcBCCPin vivo verified that this growth phenotype is due to a directinhibition of hetACCase activity by BADC3.

Example 9

Recombinant BADC Inhibits Plant hetACCase Activity

To test if the BADCs can also inhibit plant hetACCase, enzyme activityassays were performed on ten-day-old A. thaliana silique extracts. Theactivity of hetACCase was monitored in vitro in ten-d-old siliques bymeasuring the incorporation of H¹⁴CO₃ into acid-stable products. A.thaliana WT Col-0 10-d-old siliques were harvested after six hours oflight exposure. In each trial, four biological replicates of threesiliques were assayed. Siliques were pulverized in homogenization buffer(20 mM TES, pH 7.5, 10% glycerol, 5 mM EDTA, 2 mM DTT, 2 mM benzamidine,2 mM PMSF, 1% Triton X-100), centrifuged at 10 k g for 15 s, and assayedwithin 5 minutes of harvest to minimize loss of hetACCase activity.Assays were performed in the presence of 10 μM haloxyfop to inhibithomomeric ACCase activity. Enzyme activity values for (−)acetyl-CoAcontrols were subtracted from (+)acetyl-CoA trials to determine the truehetACCase activity levels. Purified recombinant protein was added toassay tubes prior to addition of silique lysate.

Assays were performed in the presence of 10 μM purified recombinantBADC1, BADC2, BADC3, BCCP2, or BSA and compared to buffer control (WT).The average of four biological replicates showed that all three BADCsinhibited hetACCase activity by 25 to 37%, while BCCP2 and BSA showed noeffect (FIG. 9). These results, in addition to the E. coli expressionresults (FIG. 8A), confirm the BADCs can negatively affect hetACCaseactivity.

Example 10

Expression Profiles of BADC and HetACCase Subunits Respond Differentlyto Light

HetACCase activity is enhanced upon light exposure in photosyntheticcells. Absolute transcript levels of the BADCs and nuclear-encodedhetACCase subunits were monitored in ten-d-old A. thaliana siliques todetermine the effect of light on gene expression. Siliques wereharvested after dark-adaption or exposure to various lengths of light.Quantitative PCR analysis of RNA extracts from these samples showed thatgene expression for each nuclear-encoded catalytic subunit to hetACCaseincreases significantly in response to light. After six hours,expression of BCCP1, BCCP2, and α-CT increased approximately 15-fold,while BC expression increased 35-fold (FIG. 10A). In contrast, BADC1 andBADC2 expression was reduced approximately ten-fold, while BADC3expression increased eight-fold. Despite the conflicting changes in BADCisoforms, total BADC transcript level was reduced by half after sixhours light exposure (FIG. 10B). The total BADC:BCCP transcript ratio isapproximately 9:1 prior to light exposure, and then shifts to almost 1:4after six hours light exposure (FIG. 10B), suggesting that BADC proteinlevels are relatively greater than BCCP protein levels in the dark, andvice versa in the light. These data further support the premise thatBADC genes are negative regulators of ACCase.

Example 11

Oil Production in Arabidopsis Thaliana Seed Increases in Response toSilencing the BADC1 Gene

The inventors further designed an in planta experiment to confirm thatsilencing BADC protein expression has a positive effect on ACCaseactivity in Arabidopsis thaliana, and results in increased fatty acidproduction. An RNAi cassette (SEQ ID NO:7) was produced and transformedinto wild type plants in order to silence BADC1 expression in A.thaliana. Inverted repeats targeting AtBADC1 were inserted into thepMU103 vector. The repeats coded for bases 774 to 1034 of the cDNAsequence (accession AT3G56130.1). Primers used to amplify the sequencewere 5′-GTGTTAGTCACATCTCCCGCAGT-3′ (SEQ ID NO:154) and5′-GATGTTGATGTCGTGGAAAGATGGC-3′ (SEQ ID NO:155). A sequence confirmedconstruct was transformed into A. thaliana ecotype Col-0 using thefloral dip method. Basta herbicide screening was used to identifyindependent lines. Expression of the RNAi cassette was driven by theglycinin promoter. For monitoring seed oil content, T2 plants from eachindependent line were grown to maturity alongside wild type plants. Dryseed was harvested for analysis.

The fatty acid content in the T3 generation were collected and analyzedto show increased oil production. Seed oil was derivatized as describedby Li et al., Phytochemistry 67, 904-915, 2006). Heptadecanoic acid wasused as an internal standard. FAMEs were analyzed by a Hewlett Packard6890 gas chromatography system. For WT and each independent line, 5 mgseed from thirteen and four plants, respectively, were analyzed. Seedswere dried over desiccant for one week prior to analysis

FIG. 11 illustrates that the oil production (in Arabidopsis thalianaseed) increases in response to silencing of the BADC1 gene. Total seedoil content of 26 independent A. thaliana lines containing the BADC1RNAi cassette has been analyzed along with wild type. The bar graphshows 22 of the 27 lines higher contained higher seed oil content onaverage. Asterisks signify statistical significance (*, P<0.05, **,P<0.01). Among the 26 lines, eight lines showed statistically higherseed oil content with increases of 7.7 to 11.7%, which is listed inTable 2.

TABLE 2 T3 lines with statistically higher seed oil content Plant line Pvalue Percent change from wild type  5-1 0.006 11.7 15-1 0.000 11.5 24-50.008 10.9 14-1 0.001 10.3  8-1 0.005 9.4 25-4 0.005 9.1 18-1 0.040 7.9 7-2 0.023 7.7

Seed oil content analysis showed a significant increase in oil in threeof six independent T2 lines (FIG. 12A). Additionally, RT-PCR analysis ofwhole silique tissue showed a significant reduction in BADC1 transcriptlevel of approximately 22% on average in the three lines containingsignificantly higher seed oil (FIG. 12B). RNA for RT-PCR and qPCRanalysis was extracted from 10-d-old siliques using the RNeasy PlantMini Kit (Qiagen). cDNA was synthesized from 500 ng RNA of fourbiological replicates. Primers used in analysis were: BADC1 sense,5′-GCTCCTAGCCCATCTCAAGC-3′ (SEQ ID NO:156); BADC1 antisense,5′-TCCAGATGCCTCCAAAGCAG-3′ (SEQ ID NO:157); Actin 8 sense,5′-CCAGATCTTCATCGTCGTGGT-3′ (SEQ ID NO:158); Actin 8 antisense,5′-ATCCAGCCTTAACCATTCCAGT-3′ (SEQ ID NO:159). qPCR assays were performedon an ABI 7500 system (Applied Biosystems). Reaction volumes were 20 μLand contained SYBR Green PCR Master Mix (Applied Biosystems). Controlreactions contained no template and were performed in triplicateAmplicon identity was confirmed through melting curve analysis. For qPCRanalysis, absolute transcript quantities were calculated using astandard curve of serially diluted amplicons of known concentrations.The fractional silencing is partly due to the use of whole siliquetissue instead of isolated seed for RT-PCR analysis. These resultsdemonstrate that BADC proteins are negative regulators of hetACCase.

Example 12

Gene Expression Levels of BADC and ACCase Genes During Seed Developmentin Arabidopsis Thaliana

FIG. 13A shows the gene expression levels of BADC and ACCase genesduring seed development in Arabidopsis thaliana (Comprehensive SystemsBiology project, csbdb.mpimp-golm.mpg.de). Although all three BADCproteins are expressed during seed filling, BADC2 showed the highestexpression over these time points. FIG. 13B shows the gene expressionlevels of BADC and ACCase genes in Glycine max during seed development(Soybase Database, soybase.org). In this species, the BADC1 ortholog isthe most highly expressed in the seed, while BADC3 is relatively lowerin expression and BADC2 is not present. Therefore RNAi silencing ofBADC1 is the most likely to increase ACCase activity in the seed ofGlycine max. Nevertheless, the presence of multiple BADC genes in mostplants suggests targeted gene silencing (i.e. specific promoter) ofmultiple copies may be necessary to obtain maximal increases in ACCaseactivity and thus oil yield, due to the potential for gene compensation.

Example 13

Dose-Dependent Inhibition of ACCase Activity by BADC1

As shown in FIG. 14, ACCase activity was monitored in vitro in20-day-old A. thaliana leaf extracts with increasing concentrations ofrecombinant BADC1. Specific activity was determined for each experimentand 0 μM controls were normalized to 1. Specific activity values forcontrols ranged from 0.84-2.00 nmol/min/mg. Each data point representsthe average of four biological replicates, with each biologicalreplicate comprised of four light-adapted leaves. The data fit to apolynomial curve where R2=0.987. The Ki for BADC1 was determined to beapproximately 4.3 μM under these conditions. SDS-PAGE analysis was alsoperformed to demonstrate purity of recombinant BADC1 protein used in theassays.

The invention claimed is:
 1. A transgenic plant transformed with arecombinant nucleic acid molecule comprising a synthetic RNAi expressioncassette that targets endogenous BADC1 gene in said transgenic plant toreduce or eliminate expression of a BADC1 protein encoded by saidendogenous BADC1 gene, wherein said synthetic RNAi expression cassettecomprises a heterologous promoter operably linked to an inverted repeatof at least 260 bp in size corresponding to a nucleotide sequenceencoding said BADC1 protein, wherein said BADC1 protein has at least 95%amino acid sequence identity to SEQ ID NO: 1, wherein said BADC1 proteinlacks the conserved biotinylation motif and biotinyl lysine residue of aBCCP1 protein, and wherein said transgenic plant exhibits (i) increasein the activity levels of the committed step for de novo fatty acidbiosynthesis, acetyl-CoAcarboxylase (ACCase) and (ii) increase in fattyacid and/or triacylglycerol production in said transgenic plant ascompared to a control plant of the same species lacking said recombinantnucleic acid molecule and grown under identical growth conditions.
 2. Atransgenic seed obtained from the transgenic plant of claim 1, whereinsaid transgenic seed has increased seed oil content as compared to acontrol seed of the same plant species, and wherein said transgenic seedcomprises said recombinant nucleic acid molecule.
 3. A method ofobtaining a transgenic plant comprising: (i) transforming plants with arecombinant nucleic acid molecule comprising a synthetic RNAi expressioncassette that targets endogenous BADC1 gene in said transgenic plants toreduce or eliminate expression of a BADC1 protein encoded by saidendogenous BADC1 gene, wherein said synthetic RNAi expression cassettecomprises a heterologous promoter operably linked to an inverted repeatof at least 260 bp in size corresponding to a nucleotide sequenceencoding said BADC1 protein, wherein said BADC1 protein has at least 95%amino acid sequence identity to SEQ ID NO: 1, and wherein said BADC1protein lacks the conserved biotinylation motif and biotinyl lysineresidue of a BCCP1 protein; and (ii) selecting a transgenic plant fromthe transgenic plants of step (i) that exhibits increase in the activitylevels of the committed step for de novo fatty acid biosynthesis,acetyl-CoAcarboxylase (ACCase) and (ii) increase in fatty acid and/ortriacylglycerol production in said transgenic plant as compared to acontrol plant of the same species lacking said recombinant nucleic acidmolecule and grown under identical growth conditions.
 4. The method ofclaim 3, further comprising obtaining transgenic seeds from the selectedtransgenic plant of step (ii), and wherein said transgenic seeds haveincrease in seed oil content as compared to oil content in control seedsof the same plant species lacking said recombinant nucleic acidmolecule.